SQUAMOUS CELL CARCINOMA ANTIGEN:

SQUAMOUS CELL CARCINOMA ANTIGEN: A NOVEL TUMOR MARKER FOR

HEPATOCELLULAR CARCINOMA

Thesis Submitted for the Partial Fulfillment of M.D. of Clinical and Chemical Pathology

BY Phebe Lotfy Abdel Messeih

MB. BCH. MSC.

Under Supervision of PROF. DR. AZZA MOHAMED ELKHAWAGA

Professor of Clinical and Chemical Pathology Faculty of Medicine – Cairo University

PROF. DR. NABIL MOSTAFA ELQADY

Professor of Tropical Medicine Faculty of Medicine – Cairo University

PROF. DR. NAGUI ABDALLA ISKANDAR

Professor of Chemical Pathology National Center for Radiation Research and Technology

Atomic Energy Authority

DR. MARIAN FATHY ISAAK Ass. Prof. of Clinical and Chemical Pathology

Faculty of Medicine – Cairo University

Faculty of Medicine Cairo University

2009

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Abstract

Serum Squamous Cell Carcinoma Antigen (SCC-Ag) by ELISA

technique and Alpha-fetoprotein (AFP) by IRMA technique were measured in

65 patients with hepatic focal lesion. 49 patients suffered from proved

hepatocellular carcinoma and 16 patients were having cirrhosis & 20 normal

controls. Median levels of serum AFP and SCC-Ag in HCC patients was

significantly higher when compared with both cirrhotic patients and controls.

On using receiver operator characteristic curve to improve sensitivity and

specificity of AFP and SCC-Ag for detection of HCC, the best chosen cut-off

values were 40 IU/mL for AFP and 2.55ng/L for SCC-Ag, these yielded a

sensitivity of 67.2% and 61.2% respectively and specificity 100%.

The diagnostic sensitivity of them increased to 87.7% when they was

combiendly calculated. It was found that the combined use of AFP and SCC-

Ag is useful in screening patients with hepatic focal lesion to increase the

chance of early diagnosis of HCC patients.

Key words:

SCC-Ag, HCC, AFP.

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To My Family

I

With Lots Of Love

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Acknowledgement

Thanks to God firstly and lastly. I feel always indebted to God, the kindest and the most merciful and who is behind all success.

In few grateful words, I would like to express my sincere thanks to all my professors who have helped me to carry out this work.

I wish, particularly, to express my deepest gratitude and appreciation to professor Dr. Azza Mohamed ElKhawaga, professor of clinical and chemical pathology, Cairo University, for her patience, continuous help, valuable advice and sincere encouragement throughout the preparation of this work.

I would like to thank professor Dr. Nabil Mostafa Elkadi, professor of tropical medicine, Cairo University, for help, guidance and continuous encouragement.

I would like to express my profound thanks to professor Dr. Nagy Abdalla Eskandar, professor of chemical pathology, National Center for Radiation Research and Technology, Atomic Energy Authority for observations, expanded experience and follow up that help this work to attain its present.

II

These words cannot express my deep appreciation and gratefulness to Dr. Marian Fathy Isaak, assistant professor of clinical and chemical pathology, Cairo University, for her friendly attitude and kind supervision in conveying her experience to complete this work.

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Words are few to express my profound thanks to Dr. Madiha Hamza Salam, assistant professor of chemical pathology, National Canter for Radiation Research and Technology, Atomic Energy Authority for honest help, her kind assistance and encouragement.

III

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Table of contents

Item Page No.

Introduction and aim of work 1 Review of literatures:

1- Liver Disease. • Anatomy of the liver • Acute Hepatitis • Chronic Hepatitis • Cirrhosis • Hepatic Tumors

2- Hepatocellular Carcinoma • Epidemiology • Etiology • Surveillance for HCC • Diagnostic evaluation of HCC • Staging of hepatocellular carcinoma • Serological tumor markers for HCC

3- Squamous Cell Carcinoma Antigen • Serpins • Genetics and Isoforms of Squamous Cell

Carcinoma Antigen • Expression pattern of SCCA isoforms • Role of SCCA in diagnosis of different disease • Squamous cell carcinoma antigen in HCC

3 4 5 7 9

13 17 20 25 32 34

42 51 63

64 67

68 71 75

Subjects and Methods 78 Results 90 Discussion 103 Summary and conclusion 108 Recommendations 110 References 111 Arabic Summary

142

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V

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List of tables

Table (1): Child-Pugh System (Page: 11)

Table (2): Classification of Hepatic Tumors (Page: 15)

Table (3): Patient Survival According to Child-Pugh Grades (Page: 42)

Table (4): Pathologic TNM Staging Sustem (Page: 44)

Table (5): Stage Grouping of TNM Classification (Page: 45)

Table (6): Okuda Classification (Page: 46)

Table (7): CLIP Classification (Page: 47)

Table (8): Gender Distribution of Subjects in the Studied Group (Page: 90)

Table (9): Age of Subjects Among the Studied Groups (Page: 91)

Table (10): Child-Pugh Score of the Patients Group (Page: 92)

Table (11): The Etiology of Cirrhosis in Group I patients (Page: 95)

Table (12): The Laboratory Results of Group Ia, Group Ib and Group II

(Page: 96)

Table (13): The Level of AFP in Group Ia, Group Ib and Group II

VI

(Page: 98)

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Table (14): The level of SCC-Ag in Group Ia, Group Ib and Group II

(Page: 99 )

Tale (15): Combined Sensitivity and Specificity for AFP and SCC-Ag

VII

(Page: 101)

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List of figures

Fig. (1): The outcome of acute hepatitis. (Page: 6)

Fig. (2): Relative incidence of the five leading cancer diagnosis worldwide.

(Page: 18)

Fig. (3): The percentage of worldwide HCC associated with either HBV,

HCV or other causes. (Page: 25)

Fig. (4): Structure of Hepatitis B Virus. (Page: 27)

Fig. (5): Ultrasound picture: Hyperechoic focal lesion (HCC). (Page: 78)

Fig. (6): Aterial phase of spiral CT full enhancement of HCC with feeding

vessel. (Page:79)

Fig. (7): Histopathlogy of HCC (grade II). (Page: 80)

Fig. (8): Histopathological findings of patients group. (Page: 89)

Fig. (9): Gender distribution of subjects in the studied group. (Page: 90)

Fig. (10): Age distribution among the studied groups. (Page: 91)

Fig. (11): Child-Pugh score of patients group. (Page: 92)

Fig. (12): Number of hepatic focal lesions in the studied groups. (Page: 93)

Fig. (13): Lesion size of hepatic focal lesion patients. (Page: 94)

Fig. (14): Etiology of cirrhoses in group I. (Page: 95)

VIII

Fig. (15): ALT and AST test results in group I and group II. (Page: 97)

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Fig. (16): Albumin level in group I and group II. (Page: 97)

Fig. (17): The Median level of AFP in the studied groups: Ia, Ib, II.

(Page: 98)

Fig. (18): The level of SCC-Ag in group Ia, group Ib and group II.

(Page: 99)

IX

Fig. (19): ROC analysis of AFP and SCC-Ag. (Page: 100)

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Introduction and Aim of work

INTRODUCTION

Hepatocellular Carcinoma (HCC) is the fifth most common cancer in the

world. Because of its increased incidence in the last decade and the expected

further increase in the next two decades, HCC is arousing great interest

(Giannelli et al., 2005).

There is rising incidence in the United States and world wide, because

HCC typically develops in patients with chronic liver disease and cirrhosis. It is

in these target populations that serum markers are most urgently needed.

(Wright et al., 2007).

Hepatocellular Carcinoma represents an important public health problem

in Egypt where up to 90% of HCC cases are attributable to hepatitis C viral

(HCV) infection. The development of effective markers for the detection of

HCC could have an impact on cancer mortality and significant public health

implications world wide. (Goldman et al., 2007).

Earlier detection of HCC can improve patient survival (Yoon, 2008).

Ultrasonography (US) and alpha-fetoprotein (AFP) monitoring are the only

reasonable screening strategy to detect HCC (Beneduce et al., 2005). AFP is the

only serological marker used in surveillance programs of hepatic patients,

although its reliability remains unsatisfactory. It is elevated in only about 60%

of HCC patients (Goldman et al., 2007). Alterations of AFP serum levels are

commonly observed in cirrhotic patients, therefore identification of new

biomarkers to establish the risk of cancer and/or detect its appearance at a pre-

clinical stage are urgently needed (Bruix and Sherman, 2005).

Squamous Cell Carcinoma antigen (SCC-Ag) is physiologically expressed

in the squamous epithelia, and increased levels have been detected in several

epithelial cancers such as those of the head, neck, cervix and lung (Suminani et

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Introduction and Aim of work

al., 1991). It is over expressed in HCC tissues and peri-tumoral tissue together

with a higher concentration in the serum of HCC patients than cirrhotic patients


AIM OF THE WORK

The aim of this study is to asses the clinical utility of SCC-Ag as a non invasive

marker for the early diagnosis of HCC.

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Review of literature

1 - LIVER DISEASES

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Anatomy of the liver

Macroscopic structure

The liver is the largest gland of the body, weighing 1200-1600 gm, it is

wedge-shaped, and covered by a network of connective tissue (Gilsson's

capsule). Situated in the upper right portion of the abdominal cavity, the liver is

divided by fissures (fossae) into four lobes: the right (the largest lobe), left,

quadrate and caudate lobes. It is connected to the diaphragm and abdominal

walls by five ligaments: the membranous falciform (also separates the right and

left lobes), coronary, right and left triangular ligaments, and the fibrous round

ligament (which is derived from the embryonic umbilical vein). The liver is the

only human organ that has the remarkable property of self-regeneration. If a part

of the liver is removed, the remaining parts can grow back to its original size

and shape. (Beuers, 1997).

Blood Supply of the liver:

The liver has double blood supply, the hepatic artery and the portal vein.

The portal vein carries blood that has already passed through the capillary bed of

the alimentary tract, and brings approximately 75% of the afferent blood that is

rich in nutrients but relatively poor in oxygen. The hepatic artery, a branch of

the celiac trunk provides the liver with about 25% of its blood supply and carries

well oxygenated blood. (Beuers, 1997).

Microscopic Anatomy:

The functional anatomic unit of the liver is the acinus, which consists of a

branch of the portal vein, hepatic artery, and bile duct. The blood vessels radiate

towards the periphery, forming sinusoids, which ultimately drain into the central

hepatic vein. (Bioulac-Sage et al., 1990).

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The liver has a limited number of ways of responding to injury. Acute

injury to the liver may be asymptomatic, but often presents as jaundice. The two

major acute liver diseases are acute hepatitis and cholestasis. Chronic liver

injury generally takes the clinical form of chronic hepatitis; its long-term

complications include cirrhosis and HCC.

Acute Hepatitis

Acute hepatitis refers to an acute injury directed against the hepatocytes.

The injury may be mediated either directly, as occurs with certain drugs such as

acetaminophen, or isoniazid, or indirectly, as occurs with immunologically

mediated injury by most of the hepatitis viruses and certain drugs (Purcell,

1994). Six viruses have been identified (A, B, C, D, E, G) as hepatotropic

viruses (Purcell, 1993). In addition, certain other viruses may infect as part of a

more generalized infection. Such as cytomegalovirus (CMV) (Dummer, 1990),

Epstein-Bar virus (EBV), and herpes simplex viruses (HSV) (Khein et al.,

1991).

In direct injury, there is typically a rapid rise in cytosolic enzymes, such

as AST, ALT, and LD, followed by a rapid fall with rates of decline similar to

known half-lives of the enzymes. With immunological injury, there is a gradual

rise in cytosolic enzymes, a plateau phase, and a gradual resolution of enzyme

elevation. Although jaundice is a key clinical finding in acute hepatitis, it is

often absent .Alkaline phosphatase ALP is usually mildly elevated, and is less

than three times the upper reference limit in 90% of cases of acute hepatitis.

Bilirubin elevation, when present, typically is predominantly direct reacting

bilirubin; indirect bilirubin is higher than direct bilirubin in about 15% of cases.

(Borsch et al., 1988).

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Diagnosis:

All forms of acute viral hepatitis have similar pathology and a similar

clinical course. They are all diagnosed on the basis of marked elevations in

aminotransferases, usually between 8 and 50 fold the upper reference limits,

with only slight elevation in ALP and little or no effect on hepatic synthetic

function. ALT is typically higher than AST because of slower clearance.

Enzyme elevations typically peak before peak bilirubin occurs, and remain

increased for an average of 4 to 5 weeks (longer for ALT than AST because of

its longer half-life). Bilirubin elevation is variable, the outcome of acute

hepatitis is variable. In most cases complete recovery occurs and liver

regeneration leads to normal structure and function. With some viruses, failure

to clear infection leads to development of chronic hepatitis. In a small

percentage of cases, massive destruction of the liver leads to acute (fulminant)

hepatic failure which is associated with high mortality unless liver

transplantation can occur (Figure 1) (Dufor. 2006).

Figure (1) The outcome of acute hepatitis (Dufor, 2006)

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Chronic Hepatitis

It is defined as a chronic inflammation of the liver that persists for at least 6

months (Garcia and Gentry, 1989). The etiology may be viral, autoimmune or

drug induced.

Viral Hepatitis:

Hepatitis B:

Approximately 10% of acutely hepatitis B virus infected people develop

chronic hepatitis, the rate is much higher in neonates and younger people

(Gitlin, 1997).

Hepatitis C:

Hepatitis C virus is the most common cause of chronic hepatitis in the

world. More than 75% of cases of acute hepatitis C become chronic, and for

most patients it is a lifetime infection (Tong et al., 1995).

Autoimmune Chronic Hepatitis:

It is an inflammatory reaction directed against the hepatic cells, and in some

cases against the bile ducts. The characteristic histological features are periportal

inflammation with piecemeal necrosis. Autoimmune hepatitis can be subdivided

into type 1 and type 2 based on specific autoantibodies which include:

antinuclear antibody (ANA). Anti-smooth muscle antibody (ASMA), anti-liver

kidney microsomal antibody type 1 (LKM1) and anti-soluble liver antibody

(cytokeratin) (Aiza and Schiff, 1995).

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Drug Induced Hepatitis:

The liver is particularly concerned with drug metabolism, especially with

drug given orally. They must be presented to the liver and converted to water

soluble compounds for excretion via the urine or bile. Some drugs can cause

toxic effects on the liver (Lee, 1995).

It is essential that any patients presenting with jaundice or altered

biochemical liver tests to be questioned carefully about exposure to chemicals at

work or at home and drug intake. (Morgan and Smallwood., 1996).

Diagnosis:

The clinical features of chronic hepatitis are highly variable. Most patients

are asymptomatic, but nonspecific features such as fatigue, lack of

concentration, and weakness may be present. Most patients are diagnosed

because of an unexplained abnormality of aminotransferases or detection of

positive results on a screening test for a cause of chronic hepatitis. Moderate

elevations of aminotransferases activities (twofold to fivefold) are characteristic,

whereas results of most other tests are normal. Normal aminotransferases

activities do not rule out histological evidence of chronic hepatitis especially in

the presence of chronic viral hepatitis or nonalcoholic steatohepatitis (NASH).

Characteristically, ALT is elevated to a greater degree that AST, although

elevations in both are common; reversal of the AST : ALT ratio to greater than 1

suggests coexisting alcohol abuse or development of cirrhosis. (Fontana and

Lok, 2002).

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Cirrhosis

Cirrhosis, defined anatomically as diffuse fibrosis with nodular

regeneration, represents the end stage of scar formation and regeneration in

chronic liver injury. This response to injury occurs independently of the etiology

and thus it is not possible, in most circumstances, to determine the cause of

cirrhosis based on the histology. Classically cirrhosis has been classified as

micronodular, macronodular, or mixed based on the histology and gross

appearance of the liver. However, this is considered inadequate for etiological or

prognostic purposes. (Dufor, 2006).

Aetiology:

Consequently, it is now more common to classify cirrhosis based on the

presumed or known etiology. The common causes of cirrhosis are:

• Viral Hepatitis B Hepatitis C

• Toxic Alcohol

• Metabolic Hemochromatosis Wilson's disease

α1-Antitrypsin deficiency

• Biliary Primary biliary cirrhosis Primary sclerosing cholangitis

• Autoimmune hepatitis

• Idiopathic

But most cases of cirrhosis occur as a result of chronic hepatitis.

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Diagnosis:

In the early stages of transition from chronic hepatitis to cirrhosis, termed

compensated cirrhosis, there may be no signs or symptoms of liver damage.

Laboratory abnormalities usually appear before clinical findings such as ascites,

gynecomastia, palmar erythema, and portal hypertension begin to develop. The

earliest laboratory abnormalities to develop in cirrhosis are: fall in platelet count,

increase in PT, decrease in the albumin to globulin ratio to less than one, and

increase in the AST/ALT activity ratio greater than one. The ratio of AST/ALT

activity is often greater than 1 in cirrhosis. The mechanism for the change in

ratio is not clear, but there appears to be a decrease in the production of ALT in

cirrhotic individuals. Increases in alpha fetoprotein (AFP) are common in

cirrhotic patients, even in the absence of HCC. (Giannini et al., 2003). In

patients presenting with ascitis, analysis of the ascitic fluid is essential.

Activities of aminotransferases are variable in cirrhosis, and reflect activity

of underlying necroinflammatory activity. If the cause of cirrhosis has been

eliminated (as by abstinence from ethanol or successful treatment of viral

hepatitis) amintransferase activity is often within the reference interval.

Persistence of elevation is a risk factor for development of HCC. (Sato et al.,

1996).

Survival in those with compensated cirrhosis is good; 10-year survival rate

in a large series was 90%. As cirrhosis progresses, decompensation occurs. A

variety of manifestations of portal hypertension may be present. Jaundice is a

late finding in decompensated cirrhosis. Once decompensation occurs 10-year

survival is only about 20%. (Luo et al., 2002).

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Severity of cirrhosis

Over the years, many clinical and biochemical parameters have been

suggested in order to predict more accurately the prognosis of cirrhotic patients

and asses correctly their short and long term survival.

The Child-Turrcote-Pugh (CTP) score is still considered the cornerstone in

the prognostic evaluation of cirrhotic patients although it was formulated more

than 30 years ago. (Pugh et al., 1973).

Currently, the model for end-stage liver disease (MELD) is introduced as a

tool to predict mortality risk and to assess disease severity in patients with

chronic liver disease so as to determine organ allocation priorities. (Kamath et

al., 2001).

Child-Pugh System:

Child-Pugh classification addresses the functional capacity of the liver

according to the degree of encephalopathy, the degree of ascites, the plasma

concentration of albumin and bilirubin and the prothrombin time (table 1).

Table (1): Child-Pugh system (Brown et al., 2002)

Feature 1 Point 2 Point 3 Point

Encephalopathy None Grade 1 – 2 Grade 3 – 4

Ascites None Slight Moderate – severe

Albumin (g/dL) >3.5 2.8 – 3.5 <2.8

Prothrombin time (s prolonged) <4 4 – 6 >6

Bilirubin mg/dL <4 4 – 10 >10

Scoring <7 points – class A; 7 – 9 points – class B; >9 points – class C.

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CTp class A patients (score less than 7 points) usually show good longer

term survival unless other events occur (for example, hepatocellular carcinoma,

uncontrolled bleeding due to portal hypertension). CTp class B patients are

considered a heterogeneous group as their clinical condition may remain stable

for more than year or rapidly deteriorate. CTp class C patients are those with

decompensated disease. They are considered the conventional candidates for

liver transplantation. (Botta et al., 2003).

Model for End-Stage Liver Disease:

The (Model for End-Stage Liver Disease) scores MELD calculated

according to the original formula proposed by Mayo Clinic group:

MELD scores = 0.957 x log (creatinine mg/dl) + 0.378 x log (bilirubin

mg/dl) + 1.120 x log (INR) + 0.643.

It appears superior to the Child-Pugh scoring system in predicting short

term survival. Risk of death over 3 months is low in those with MELD scores

below 10, intermediate in those with scores of 10 to 20, and high in those with

scores above 20. (Forman and Lucey, 2001).

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Hepatic Tumors

The liver is host to a wide variety of both benign and malignant primary

tumors. It is also the second most common site of metastasis. Metastatic tumors

account for 90% - 95% of all hepatic malignancies. The liver size, anatomical

location, dual blood supply and the availability of nutritional material are all

factors that influence the deposition and growth of the neoplasm. The primary

tumors may arise from many cell lines in the liver but most commonly from

parenchymal and biliary epithelial cells and mesenchymal cells. The two most

important primary liver tumors are hepatocellular carcinoma and

cholangiocarcinoma (table 2). (Dufor, 2006).

Benign Tumors:

The most common benign hepatic tumor is haemangioma, occurring in at

least 1% of the population however, other rare tumors may occur as

hepatocellular adenoma which occurs largely in females. It's incidence has

increased in the last few decades, probably related to the introduction and

increased use of oral contraceptives.

Other rare benign tumors include: Bile duct adenoma, cystadenoma,

fibroma, liomyoma. (Di-Bisceglie, 1998).

Malignant Tumors:

Malignant tumors include: primary liver tumors and metastatic lesions.

Metastatic lesions (secondary deposits in the liver) are much more common than

primary liver tumors (20:1). The importance of the liver as a common target for

metastatic malignant cells is mainly due to its dual blood supply and about 25%

of the cardiac output it receives every minute. Nearly one third of all cancers

ultimately spread to the liver (Jarell and Carabasi, 1990). The most common

sources include: Colon, Pancreas, Stomach, Breast, Bronchogenic. (Dufor,

2006).

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Metastasis reach the liver by one of the following routes:

• Direct spread: from the stomach, gall bladder, and colon.

• Blood spread: via

- Portal vein: from the stomach, colon, rectum, and the head of

pancreas.

- Hepatic artery: from the lungs, breast, kidney, and leukemia.

• Lymphatic spread: from the lung and breast. (Rifaat, 1998).

Primary malignant liver tumors include epithelial and mesenchymal

tumors. Hepatocellular carcinoma is primary epithelial liver tumor. It is the fifth

most common cancer world wide occurring mainly on top of infection with

HBV or HCV. Once cirrhosis has developed, the rate of development of HCC is

about 1.5% - 5% per year in both HBV and HCV patients. (Fattovich et al.,

1997).

Cholangiocarcinoma accounts for 10 – 20% of primary liver tumors and

arises form bile duct epithelium.

Other rare primary malignant tumors include:

Cystadenoma, squamous carcinoma and hepatoblostoma. (Lawrence, 1996).

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Table (2): Classification of hepatic tumors (Dufor, 2006)

Hepatic Tumor

Liver Cell Line Benign Malignant

Epithelial

Adenoma

Bile duct adenoma

Cystadenoma

Carcinoid

Focal nodular hyperplasia

Diffuse nodular hyperplasia

Hepatocellular carcinoma

Colangiocarcimona

Cystadenocarcinoma

Squamous carcinoma

Mesenchymal

Cavernous hemangioma

Fibroma

Leiomyoma

Hematoma

Hemangiosarcoma

Fibrosarcoma

Liomyosarcoma

Hepatoblastoma

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Tumor Like Lesion Of The Hepatocytes:

These include: Focal nodular hyperplasia (FNH), Nodular regenerative

hyperplasia (NRH), Adenomatous hyperplasia and inflammatory Pseudotumor.

Focal Nodular Hyperplasia (FNH):

It represents an abnormal proliferation of hepatocytes around an abnormal

hepatic artery which is usually embedded in a characteristics central stellate scar

(Kerlin et al., 1983).

Nodular regenerative hyperplasia (NRH):

Is characterized by the diffuse formation of nodules comprised of

hepatocytes throughout the liver. This is similar to cirrhosis except that these

nodules do not have a surrounding rim of fibrosis (Stromeyer and Ishak, 1981).

Adenomatous hyperplasia (Macro-Regenrative nodules):

It is a term used for regenerative nodules of hepatocytes greater than 1cm

in diameter found in association with cirrhosis or rarely in submassive hepatic

necrosis (Di-Bisceglie, 1998).

Inflammatory pseudotumor:

It is an area of chronic inflammation and fibrosis. It may cause pain and

fever (Di-Bisceglie, 1998).

Other lesions of the liver:

Liver abscess, amoebic abscess, hydatid cyst, simple hepatic cyst, and

focal fatty infiltration: all are benign focal hepatic masses which are not true

tumors. Their significance lies in that they must be considered in the work up for

the evaluation of a focal hepatic masses. (Lawrence, 1996).

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2- Hepatocellular Carcinoma

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Hepatocellular Carcinoma

Hepatocellular Carcinoma (HCC) is the fifth most frequent cancer,

worldwide, with an estimate of more than 500000 incidences in year 2000.

As shown in the figure:

The relative incidence of the five leading cancers worldwide in a

descending manner are as follows: Lung cancer then breast cancer followed by

colorectal & stomach cancer, the fifth is cancer liver (Figure 2) (Parkin et al.,

2001)

Figure (2): Relative incidence of the five leading cancer diagnosis,

worldwide. (Parkin et al., 2001).

Despite being fifth in cancer incidence, worldwide, HCC is the

third leading cause of cancer death. The high mortality associated with HCC is

because it is often unresponsive to treatment. The high mortality may be in part

because the noncapsular part of the liver is lacking sensory fiber and symptoms

of HCC often occur late HCC and with a 5 year survival rate of less than 5%

with or without therapeutic intervention (El-Serag et al., 1999).

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Removal of HCC by surgical means offers the best chance for

possible cure. Criteria for such intervention have been refined over the last

decade to optimize long-term survival in selected patients. Unfortunately <20%

of patients meet the criteria for resection at time of diagnosis, so that the life

expectancy of patients with HCC is poor, with a mean survival of 6 – 20

months. (Bismuth et al., 1999).

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Epidemiology

Geographic Distribution:

Incidence rates demonstrate dramatic geographic variability ranging from

less than 5 new cases per 100000 persons per year in developed western

countries to more than 100 per 100000 persons per year in parts of south-east

Asia and sub-saharan Africa. (Pal and Pande, 2001).

The epidemiology of HCC exhibits two main patterns: one in North

America and Western Europe and another in non-Western countries, such as

those in sub-Saharan Africa, central and Southeast Asia, and the Amazon basin.

Non-Western Countries

In some parts of the world-such as sub – Saharan Africa and Southeast

Asia – HCC is the most common cancer, generally affecting men more than

women, and with an age of onset between late teens and 30s. this variability is in

part due to the different patterns of hepatitis B transmission in different

populations-infection at or around birth predispose to earlier cancer than if

people are infected later. The time between hepatitis B infection and

development into HCC can be years even decades.

North America and Western Europe

Most malignant tumors of the liver discovered in western patients are

metastasis (spread) from tumors elsewhere. In the West, HCC is generally seen

as rare cancer, normally of those with pre-existing liver disease. It is often

detected by ultrasound screening, and so can be discovered by health care

facilities much earlier than in developing regions such as Sub-Saharan Africa.

(Robbins and Cotran, 2003).

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Sex distribution:

Hepatocellular carcinoma is more common in males than females with the

ratio 4:1. This ratio varies widely according to the geographic distribution and

may reach up to 8:1.

Age distribution:

The peak age incidence of hepatocellular carcinoma varies from 40 years

in Southern Africa, 40 – 60 years in Asia and 80 years in the UK. (Zaman et al.,

1985).

Racial distribution:

In mixed population, certain races have higher incidence of the

hepatocellualr carcinoma particularly the black population of South Africa and

the ethnic Chinese in South – East Asia. (Zaman et al, 1985).

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Hepatocellular Carcinoma in Egypt:

The burden of hepatocellular carcinoma has been increasing in Egypt with a

doubling in the incidence rate in the past 10 years. This has been attributed to

several biological (eg. Hepatitis B and C virus infection) and environmental

factors (eg. Aflatoxin). Other factors such as cigarette smoking, occupational

exposure to chemicals such as pesticides, and endemic infections in the

community, such as schistosomiasis, may have additional roles in the etiology or

progression of the disease. Estimates of the burden of cancer caused by these

factors provide an opportunity for prevention. (Anwar et al., 2008).

The role of exposure to aflatoxins and development of HCC in Egypt was

historically less clear. Nevertheless, recent food sampling surveys and

population-based studies indicated that exposure to aflatoxins in Egypt may

have been underestimated in the past. Recent results indicated that both local

and imported samples were positive for aflatoxin B1 (AFB1, 17.5% and 20%,

respectively), with concentrations ranging from 3 to 25 µg/kg. The level of

AFB1 was dependent on the area of collection as well as the season of the year.

(Anwar et al., 2008).

Schistosomiasis was traditionally the most important public health

problem and infection with Schistosoma mansoni the major cause of liver

disease. From the 1950s until the 1980s, the Egyptian Ministry of Health (MOH)

undertook large control campaigns using intravenous tartar emetic, the standard

treatment of schistosomiasis, as community-wide therapy. This commendable

effort to control major health problem unfortunately established a very large

reservoir of hepatitis C virus (HCV) in the country. By the mid 1980s, the

effective oral drug, praziquantel, replace tartar emetic as treatment for

schistosomiasis in the entire country. This both reduced schistosomal

transmission and disease and interrupted the "occult" HCV epidemic. It was

evident when diagnostic serology became available in the 1990s that HCV had

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replaced schistosomiasis as the predominant cause of chronic liver disease.

Epidemiological studies reported a high prevalence and incidence of HCV,

particularly within families in rural areas endemic for schistosomiasis. Clinical

studies showed 70% to 90% of patients with chronic hepatitis, cirrhosis, or

hepatocellular carcinoma had HCV infections. Co-infections with

schistosomiasis caused more severe liver disease than infection with HCV alone.

Schistosomiasis was reported to cause an imbalance in HCV-specific T-cell

responses leading to increased viral load, a higher probability of HCV

chronicity, and more rapid progression of complications in co-infected persons.

As complications of HCV usually occur after 20 years of infection, the peak

impact of the Egyptian outbreak has not yet occurred. Efforts have been initiated

by the Egyptian MOH to prevent new infections and complications of HCV in

the estimated 6 million infected persons. (Strickland, 2006).

There was strong evidence that hepatitis B virus (HBV) was the major

cause of HCC in Egypt, but more recently HCV has become the predominant

factor associated with the more recent epidemic of HCC. It has been well

documented that Egypt has one of the highest prevalence rates of HCV infection

and disease progression, however, are influenced by additional factors such as

duration of infection, age at infection, sex, co-infection with HBV, the level of

HCV viraemia and its genotype. (Anwar et al., 2008).

In a single center experience, they reviewed the medical records of all

patients attending Cairo Liver Center during the years 1992 – 1995 to determine

the sociodemographic characteristics and they reported that HCC accounted for

4.7% (321/6850) of chronic liver diseases in patients included in their study.

HCV Ab positive cases were strikingly high (71.1%) and HBs Ag positive cases

were reported in 22.4% of patients. There was an annual significant rise of HCC

ranging from 3.6% in 1992 to 5.3% in 1995. (El-Zayadi et al., 2005).

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The number of HCC patients increased yearly from only 9 patients

evaluated at 1992 to 80 patients in the first 5 months of the year 2005. The mean

age was 54.26+/-9.2%, with high prevalence between 51 and 60 years. Male to

female ratio was 5:1. HCC is commonly diagnosed at an asymptomatic phase by

routine ultrasound (US) or because of sudden worsening of the underlying

cirrhosis. (Abdel-Wahab et al., 2007).

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Etiology

The majority of cases of hepatocellular carcinoma (HCC) in the world are

due to hepatitis B virus (HBV), with the number of hepatitis C virus (HCV)

associated cases growing in the western world and the incidence of non viral

HCC is also rising in the united states (Parkin et al., 2001) Figure 3. Non viral

causes include: cirrhosis, schistosomiasis ; aflatoxin exposure and radiation

(Parkin et al., 2001). However, HCV is the major cause of HCC in Egypt

(Anwar etal., 2008).

Figure (3): The percentage of world wide HCC associated with either

HBV, HCV or other causes (Parkin et al., 2001).

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Viral Hepatitis:

Natural history of HBV and HCV infection

Both HBV and HCV infection in people is characterized by ability to

cause either acute infection that is frequently clinically inapparent or an

unresolved, long-term persistence. In either outcome, the liver is the primary site

of replication. "Acute" infections of adults are usually inapparent, although in

some cases (perhaps fewer than 1%) a severe life-threatening hepatitis occurs.

As implied, for both HBV and HCV, symptoms associated with long-term

(chronic) infection may not be apparent for years but eventually present as

fatigue, malaise and other conditions typical of hepatitis (Lok et al., 2001).

Cirrhosis and primary liver cancer, as mentioned, often follow, and may

be the results of many years of unresolved persistent infection. The rate at which

HCC occurs in the individual chronically infected with HBV or HCV associated

cirrhosis is between 1 and 6% per year. (El Serag et al., 2001).

Virology of HBV

HBV is a member of a virus family called "Hepadnaviridae" and is small,

partially double-stranded DNA ( ~3.5 kb). As shown in figure 4, its specifies a

small number of known gene products, including a reverse transcriptase/DNA

polymerase (pol), capsid protein (core), envelope (env) proteins (L, M and S) as

well as proteins of uncertain function such as 'X' and 'e'. (Li et al., 2006).

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Figure (4): Structure of the hepatitis B virus (Block et al., 2003).

The HBV genome is shown in a circular form, with bold lines

representing transcripts corresponding to the env (envelope or surface),

polymerase, 'X', and core products. The numbers indicate the nucleotide

positions using the EcoR1 site as an arbitrary beginning. The nucleotide

positions of the AUG (first codons) of envelope polypeptides PreS1, PreS2 and S

are shown. (Block et al., 2003).

Viral oncology of HBV:

For most HBV-induced HCC, the molecular mechanisms are unknown.

The 154-aminoacid viral gene product 'X' is probably the viral function most

frequently implicated in oncogenesis. It is named 'X' because of uncertainty

about its function. With regard to oncogenesis, X can inactivate or complex with

the cellular antioncogene product, p53, which is frequently disabled in HCC

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(Feitelson, 1999). This activity alone could account for any cellular

transformation properties. However, p53 inactivation may occur in only a

minority of HBV-induced HCCs, and the relevance of the X: antioncogene

observations, however logical, controversial. (Zoulim et al., 1994).

The hepatitis B virus (HBV) X protein variants encoded by HBV

genomes found integrated in genomic DNA from liver tumors of patients with

HCC generally lacks amino acids 134 to 154 (Li et al., 2006). The X gene

product has been shown to transactivate HBV promoters as well as cellular

functions associated with cellular growth such as c-fos, c-jun, c-myc and EGF.

X function has also been implicated in influencing DNA repair. (Yeh, 2000).

Cytologically, overproduction of viral envelope proteins, particularly L

and possibly M, results in their intracellular accumulation and may predispose

the cell to stress, which in turn may lead to the development of cancer. In

addition, HBV envelope protein mutants that overaccumulate envelope

polypeptides within the cell have been observed to be associated with advancing

liver disease and may be in part, responsible for ground glass hepatocytes and

perhaps even HCC lesions (Tai et al., 2002). HBV replication appears to involve

heat shock proteins that could lead to a cell response that involve mutagenic

reactants. (Wu et al., 2002).

Viral Oncology of HCV:

The pathogenesis of HCC from HCV infection is yet to be fully

understood, with various viral protein – host cell interactions hypothesized to

play a direct role in the development of HCC. Perturbations in the cell cycle,

combined with upregulation of oncogenes and loss of tumour-suppressor gene

functions, may combine to lead to HCC development; HCV proteins have been

shown to interact with these cellular pathways. The natural history of HCV

infections is progression to fibrosis and cirrhosis, leading to HCC in a

significant proportion of the infected population. These viral protein – host cell

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interactions may play a role separate from cirrhosis in the development of HCC

but they also play an integral role in the cause of chronic infection, leading to

fibrosis and cirrhosis. (Tran, 2008).

The core protein is able to bind to p53, a key component in cell cycle

arrest and apoptosis. By allowing HCV to modulate the cell cycle, it can either

prevent apoptosis and allow the replication of its progeny or induce apoptosis to

enable viral spread. The disruption of checkpoints in various stages of the cell

cycle is one way that tumourigenesis can occur. Core may enhance p53 function

by increasing the affinity of P53 to its DNA-binding site or by increasing its

transcriptional activity without increasing p53 expression itself. However,

although a small proportion of core is found in the nucleus, core is

predominantly cytoplasmic and p53 is located within the nucleus, which may

suggest that p53 enhancement may not be fully explained by these mechanisms.

(Otsuka et al., 2000).

Liver cirrhosis

There is a high prevalence of cirrhosis in patients with HCC (between 60

– 90%). There is an increased risk of patients with cirrhosis to develop HCC.

The risk is greater with macronodular cirrhosis that is commonly caused by

HBV infection. Micronodular cirrhosis caused most often by alcohol abuse is

complicated by carcinoma between 3 – 10% of cases (Weinberg et al., 1976).

HCC is rare in the other varieties of cirrhosis. (Guan et al., 1985). Cirrhosis

associated with viral hepatitis generally leads to HCC more readily than non-

viral-induced cirrhosis.

Cirrhosis, however, is not a prerequisite for HCC. As many as 30% of

chronic hepatitis B patients who develop HCC are non-cirrhotic. In one study

from France, 25% of patients who underwent surgical resection of HCC had

either minimal or no cirrhosis. (Bralet et al, 2000).

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Schistosomiasis:

Bilharzial toxins might lead through cirrhosis and other complications of

hepatic bilharziasis to the regeneration of liver nodules, which may lead to

mutation and liver malignancy. It was proved that schistosomial liver disease

might alter the metabolism of enviromental mutagens such as aflatoxin.

However, a combination of schistosomiasis and carcinogens, result in marked

liver cancer rather than one of them alone (Sherlock, 1989).

Aflatoxins:

Aflatoxins, secondary metabolites produced by Aspergillus flavus and

Aspergillus parasiticus are potent human carcinogens implicated in HCC

(Murugavel et al., 2007).

The prevalence of aflatoxin B1 (AFB1) as co-carcinogen was analyzed in

liver biopsies and liver resection specimens from histopathologically proven

HCC patients and in the control group (biopsy from cirrhosis patients). Serum

samples were tested for aflatoxin B1 using an ELISA kit. In spite of positive

AFB1 immounostaining in HCC cases, all serum specimens from both HCC and

the control group were AFB1 negative. Thus AFB1 staining was significantly

associated with tumor tissue.

The impact seems to be a cumulative process, as revealed by the AFB1

deposits in HCC liver tissue, even though the serum levels were undetectable

(Murugavel et al., 2007).

AFB1 itself is harmless but it is metabolized in a phase 1 reaction to a

reactive metabolite AFB 8,9-epoxide which is mutagenic and binds to guanosine

bases in DNA and G to T transvertion occurs, Codon 249 of p53 is a target for

this reaction in which this tumour suppressor gene becomes inactivated. AFB

8,9 epoxide is rendered harmless by glutathione S transferase that converts it to

glutathione conjugate which is metabolized to 1,2 dihydrodiol by epoxide

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hydrolase, however, both detoxifying enzymes are polymorphic and the mutant

forms are less active, patients with HCC are likely to have the mutagenic forms

causing accumulation of 8,9 epoxide. (Sylla et al., 1999).

Rare Causes

Radiation

Recent studies of atomic bomb survivors have shown that risks of liver

cancer are significantly increased by radiation exposure. This contrast with

mortality studies of other radiation – exposed populations, which generally have

not shown a significant radiation effect for this cancer,

Liver cancer was consistently associated with radiation exposure in

studies of four cohorts exposed to thorotrast, a previously used radiology

contrast agent. However, the histologic subtypes of liver cancer and type of

radiation exposure differ from those experienced by the A-bomb survivors.

Liver cancer in atomic bomb survivors is primarily hepatocellular carcinoma

(HCC), rather than the cholangiocarcinoma and hemangiosarcoma subtypes

more associated with thorotrast exposure (Sharp et al., 2002).

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Surveillance for Hepatocellular Carcinoma

Identification of early HCC which is potentially amenable to aggressive

intervention and improved survival is the rationale behind screening for HCC.

An effective screening program, however, requires certain criteria to be

successful, including the following: a common disease with substantial

mortality, an identifiable target group, acceptable tests with high sensitivity and

specificity, and available treatment. (Prorok, 1992).

Surveillance of individuals at risk for HCC has been a matter of

controversy for decades. Geographic variations in target populations, screening

tools, and therapy complicate assessment of international literature on the

effectiveness of surveillance for HCC.. To date no substantial evidence has

accumulated which improves survival benefit with surveillance of high-risk

patients. As a result no universally accepted guidelines are available. Several

large studies on surveillance to suggest benefits. (Tong et al., 2001).

Bolondi et al. 2001, demonstrated a median survival of 30 months in

patients whose HCC was detected by surveillance versus 15 months in those

discovered by chance.

Surveillance intervals for HCC are based on balance between the tumor

doubling time and the cost of the screening tests. Most study protocols conduct

screening every 6 months. The overall cost of surveillance for HCC varies

according to region, population incidence, and the screening tools used. (Koteish

and Thuluvath, 2002).

Long-term survival requires detection of small tumors, often present in

asymptomatic individuals, which may be more amenable to invasive therapeutic

options. Surveillance of high-risk individuals for HCC is commonly performed

using the serum marker Alfa-fetoprotein (AFP) often in combination with

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ultrasonography. Various other serologic markers are currently being tested to

help improve surveillance accuracy (Bialecki and Di Bisceglie, 2005).

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Diagnostic Evaluation of HCC

Diagnosis of HCC often requires sophisticated imaging modalities such as

CT scan and MRI, which have multiphasic contrast enhancement capabilities.

Serum AFP used alone can be helpful if levels are markedly elevated, which

occurs in less than half of cases at time of diagnosis. Confirmation by liver

biopsy can be performed under circumstances when the diagnosis of HCC

remains unclear (Bialecki and Di Bisceglie, 2005).

Clinical Presentation of HCC:

HCC classically arises and grows in silent fashion, making its discovery

challenging prior to the development of later stage disease. The various clinical

presentations are generally related to the extent of hepatic reserve at time of

diagnosis, cirrhotic patients frequently present with non-specific signs and

symptoms of hepatic decompensation such as jaundice, hepatic encephalopathy,

and anasarca. Ascites, variceal bleeding or other findings consistent with portal

hypertension may indicate malignant invasion of HCC into portal structures.

Abnormal laboratory values are nonspecific for chronic liver disease (Schafer

and Sorrell, 1999).

Noncirrhotic patients with HCC typically present in a different manner, as

is commonly seen in sub-Saharan Africa and other high incidence areas. Their

tumors are often allowed to grow with much less restriction. Symptoms are

often related to long-standing malignancy and tumor growth including malaise,

anorexia, wasting, right upper quadrant abdominal pain, and distension.

(Schafer and Sorrell, 1999).

Physical examination may reveal an abdominal mass or hepatomegaly

with hard and irregular borders that may demonstrate a vascular bruit. Painless

obstructive jaundice can indicate tumor encroachment on to adjacent

extrahepatic biliary structures (Murata et al., 2003).

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Extrahepatic manifestations of HCC are well described and may relate

either to distant metastasis or paraneoplastic phenomena. Advanced HCC can

metastatasize to any organ system via hematogenous or lymphatic routes, and

most commonly spreads to bone, lung, and abdominal viscera. Paraneoplastic

manifestation occur rarely in HCC and include hypoglycemia, hypocalcemia,

polycythemia, and feminization syndrome. (Luo, 2002).

Watery diarrhea has been shown to be significantly more common with

cirrhosis and HCC than with cirrhosis alone and can be initial presenting

symptom. Increased production of intestinal secretory substances, such as

gastrin and vasoactive intestinal peptide (VIP), has been suggested as a possible

cause (Bruix et al., 1990).

Diagnostic Imaging

Imaging plays a key role in the diagnosis of HCC. Advances in imaging

technology over the past two decades have contributed to better characterization

of hepatic lesions with a wider array of options. Regardless, detection of small

tumors continues to be difficult, particularly in cirrhotic individuals whose

parenchymal architecture is abnormal. Differentiating HCC from benign lesions

commonly seen in cirrhosis or from secondary malignancies remains a

challenge.

Ultrasound (US)

US imaging has largely been replaced in diagnosis by Computerized

Tomography scan (CT) and Magnetic Resonance Imaging (MRI) as a diagnostic

instrument of choice as a result of low sensitivity and positive predictive value

with coexisting cirrhosis. The recent addition of sonographic contrast agents

such as intraarterial carbon dioxide and helium shows promise in improving

accuracy (Choi et al., 2002). However, application of duplex and color Doppler

sonography can be particularly useful in the assessment of intraheptic vascular

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flow. HCC lesions typically display fine branching patterns of increased

vascularity with greater flow velocity than metastatic lesions or hemangiomas.

(Reinhold et al., 1995).

Doppler evaluation of the portal vein can help to differentiate thrombus

from tumor invasion. Malignant portal invasion commonly produces wave forms

demonstrating arterial flow. The power Doppler is thought to be three to five

times more sensitive in detecting tumor vascularity than color Doppler by

eliminating angle dependence. (Koito et al., 1998).

Computerized Tomography Scan (CT)

CT evaluation of patients with suspected HCC should be done using

multiphasic contrast imaging of liver. Following rapid intravenous infusion of

contrast, imaging is conducted at various time intervals corresponding to the

phase of contrast enhancement. Triphasic scanning denotes hepatic imaging

performed before contrast, during arterial and venous phases. HCC tumors

derive blood flow predominantly from the hepatic artery and tend to enhance

during the arterial phase or 2 – 40 seconds after contrast infusion. The

surrounding hepatic parenchyma obtains 75 – 80 % of its blood flow through the

portal vein and is best demonstrated 50 – 90 seconds after infusion of contrast

during the portal phase. Arterial phase enhancement can increase HCC tumor

detection by 10% . (Baron et al., 1996).

HCC typically appears heterogeneous on CT, which may reflect

intratumoral fibrous standing (mosaic sign), fatty metamorphosis, necrosis, or

calcifications (Stevens et al., 1996).

The presence of satellite nodules in close proximity to the lesion is often

characteristic. Fibrous structures within or encapsulating the lesion strongly

retain contrast and enhance readily on delayed imaging (3 – 10 min after

infusion). (Yoshikawa et al., 1992).

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Brancatelli et al., 2003 described hepatic lesions which can mimic HCC

on CT imaging including regenerating nodules, hemangiomas, focal fat and

dysplastic nodules. The accuracy increases with greater imaging speed, which

allows faster administration of contrast media, thereby dramatically improving

contrast enhancement. (Choi, 2004).

The added speed and flexibility of multidetector CT (MDCT) allows high

quality, thin-section imaging with three-dimensional capabilities. (Szklaruk et

al., 2003).

CT arteriography is a more invasive yet effective option to improve

accuracy as a result of higher quantity of contrast administered at a faster rate. In

a large population-based study, Oliver et al., 1997 reported a 66% increase in

detection of HCC foci compared with triphasic CT scanning. However, the

invasive and costly nature of this approach tends to restrict its use. CT

arteriography appear to be used more often in the Far East to define hepatic

vasculature before surgical intervention.

Magnetic Resonance Imaging (MRI):

MRI uses similar concepts to those applied to CT imaging when

evaluating hepatic lesions suspicious for HCC. Recent advances in MR

technology allow images to be obtained within the time frame of one breath

hold. T1- and T2-weighted sequence images of HCC lesions vary considerably

but typically appear hypointense and hyperintense, respectively. Focal

hemorrhage, fatty change, or tumor accumulation of copper and glycogen

contribute to this inconsistency. MRI sensitivity is lowest when evaluating

tumor <2cm in diameter. (Krinsky et al., 2001).

Dynamic gadolinium contrast imaging enhances arterial blood supply

during the early phase, which improves characterization of HCC tumors. The

sensitivity and specificity are similar to those of multiphasic CT scan imaging.

The addition of superparamagnetic iron oxide contrast has been investigated to

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improve accuracy, particularly with T2-weighted sequencing. (Sadek et al.,

1995).

Superparamagnetic iron oxide comprises tissue specific MRI contrast

agent particles taken up by Kupffer cells in the liver. The combination of

superparamagnetic iron oxide-enhanced and gadolinium chelate-enhanced

dynamic MRI produces results comparable to those of CT hepatic arteriography.

MRI has become the diagnostic imaging mode of choice for HCC at many

institutions worldwide. (Yu and Kim, 2002).

Liver Biopsy:

Diagnostic evaluation of hepatic lesions with liver biopsy has been

practiced for over half a century. When performed at specialized centers, liver

biopsy offers a safe and effective means to confirm suspicious lesions for HCC.

Cytologic and histologic samples can be obtained by percutanous fine-needle

aspiration (FNA) and needle core biopsy, respectively, under US or CT

guidance. The diagnostic accuracy of liver biopsy is greater when both FNA and

core biopsy techniques are used simultaneously than when either is used alone.

The sensitivity and specificity are superior to any other diagnostic test, at 96%

and 95% respectively. (Borzio et al., 1994).

An on-site pathologist can provide immediate interpretations of cytologic

cell blocks to assure proper placement of the biopsy needle. Open surgical

biopsy procedures may sometimes be performed when suspected HCC lesions

cannot be accurately located by radiographic methods. Microscopic features of

HCC include elevated nuclear to cytoplasmic ratio, trabecular architecture,

atypical naked nuclei, and peripheral endothelial wrapping. (Pitman, 1998).

Histologic appearance ranges from nearly normal-appearing hepatocytes

in well differentiated tumors to the largely anaplastic multinucleate giant cells

characteristics of poorly differentiated HCC. (Robbins et al., 1987).

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Tumor grade was scored using the modified nuclear grading scheme

outlined by the Edmondson and Steiner, with tumor grade categorized as low,

intermediate, or high. Specifically, modified Edmondson-Steiner grades 1 and 2

were defined as well-differentiated, grade 3 as moderately differentiated, and

grade 4 as poorly differentiated. In all cases, tumor grade was defined by the

poorest degree of differentiation identified within the tumor upon pathologic

analysis of the entire specimen. (Edmondson and Steiner, 1954).

Distinguishing well differentiated HCC from benign hepatic masses such

as adenoma or focal nodular hyperplasia may be difficult. The most

recognizable premalignant histological finding is dysplasia. Liver biopsy need

not be performed under circumstances in which the diagnosis of HCC is certain

after clinical, laboratory, and radiographic evaluation. Confirmation of HCC

with liver biopsy plays a larger role in various other emerging scenarios. One

such scenario is prior to orthotopic liver transplantation or hepatic resection.

Routine surveillance programs are more frequently identifying tumors in

younger asymptomatic patients with smaller lesions and better hepatic reserve.

Many of these patients are eligible for surgical interventions which can

significantly improve survival. (Inoue et al., 2004).

Without preoperative confirmation of HCC by liver biopsy several studies

have shown that the rate of false-positive diagnosis can be substantial in patients

with small tumors. (Caturelli et al., 2002).

Hayashi et al., 2004 recently demonstrated that the false-positive rate can

be as high as 33% after histological examination of the explants. This risk of

subjecting patients with small hepatic lesions to unnecessary surgical

intervention can be limited by performing liver biopsy. The accuracy of liver

biopsy in diagnosing lesions <2cm in diameter is 95.6%. Complications

associated with liver biopsy are rare and can be diminished by using a one stick

approach, such as the coaxial technique. Mortality rates are between 0.006% and

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0.3%, with risk of serious hemorrhage or infection <1%. Liver biopsy should be

avoided when platelet counts are <50 000 per mm3 or the international

normalizing ratio (INR) is >2.

The potential for spread of tumor from the biopsy needle track is of great

concern and fuels much of the controversy surrounding the need for liver

biopsy. Although several studies show rates as high as 5%, the majority of large

studies indicate that the risk is closer to 1%. (Ohlsson et al., 2002).

Another common scenario in which the liver biopsy can be useful is in the

patient whose suspicious lesion does not necessarily meet the characteristic

radiographic or laboratory features of HCC. For example, the patient with an

AFP <400 µg/ml with a lesion which fully demonstrates arterial enhancement on

multiphasic CT imaging. AFP <400 µg/ml can be present in as many as 60% of

patients at presentation, while HCC tumor with fatty change or necrosis can

impede characteristic radiographic arterial enhancement. Many patients fit this

"gray zone" and confirmation of the diagnosis is important, especially as some

will go on to have interventions such as radio-frequency ablation, transarterial

chemoembolization, or chemotherapy (Bialecki and Di-Bisceglie, 2005).

The diagnosis of HCC poses many challenges which can vary among

different regions and centers. AFP and US imaging are most often used every 6

months for surveillance purposes in high-risk individuals. In the presence of

rising AFP or suspicion of underlying malignancy, surveillance intervals should

be shortened and more sensitive imaging techniques such as multiphasic CT

scan or MRI can be applied. The intensity of diagnostic work-up should be

individualized and tailored according to each patient's potential to tolerate

aggressive therapeutic interventions. Liver biopsy can confirm diagnosis when

necessary or rule out other lesions that may mimic HCC. Several diagnostic

strategies have been proposed, many of which are center-and region-specific.

The European Association for the Study of the Liver (EASL), for example, has

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listed standard criteria for diagnosis of HCC that incorporate both invasive and

noninvasive measures. Noninvasive criteria include two imaging techniques,

both demonstrating a focal lesion >2cm in diameter with features of arterial

hypervascularization, or a single radiologic study with these features combined

with a serum AFP level of >400 µg/ml. Use of this and other criteria can be very

helpful, but the lack of evidence-based studies should preclude their strict use in

diagnosis of HCC. Further research studies continue to focus on developing

ways to improve diagnostic tools and strategies with the aim of identifying

earlier stages of HCC. (Bialecki and Di-Bisceglie, 2005).

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Staging of Hepatocellular Carcinoma

Cancer staging should serve to select the appropriate primary and

adjuvant therapy, to estimate the prognosis, and also to assist in the evaluation

of the results of treatment. (Fleming, 2001).

Child-Pugh Classification

Initially, the Child-Pugh scoring system was used for identification of

HCC candidates for therapy. However, the Child-Pugh classification only

addresses the functional capacity of the liver without including any tumor

parameters. (Pugh et al., 1973).

Child-Pugh classification of severity of liver disease, addresses the

functional capacity of the liver according the degree of ascites, the plasma

concentrations of bilirubin and albumin, the prothrombin time and the degree of

encephalopathy, as shown in (Table 1). A total of score of 5 – 6 is considered

grade A (well-compensated disease); 7 – 9 grade B (significant functional

compromise); and 10 – 15 is grade C (decompensated disease). These grades

correlate with one – and two – year patient survival (Table 3)

Table (3): Patient survival according to Child-Pugh grades (Pugh et al., 1973)

Grade Points One-year patient

survival (%)

Two-year patient

survival (%)

A: Well-compensated

disease 5 – 6 100 85

B: Significant functional

compromise 7 – 9 80 60

C: Decompensated disease 10 – 15 45 35

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Pathologic Tumor Node Metastasis System:

The current system for staging HCC is the pathologic TNM (pTNM)

staging system. The stated goals of TNM system are: to aid the clinician in the

planning of cancer treatment, to give some indications of prognosis, and to assist

in the evaluation of treatment results (Marsh et al., 2000).

In the TNM system (Tables 4 and 5), the primary lesion is defined by

tumor size, the number and location of lesions, invasion of vascular structures

and the biliary extension. In addition, this staging system addresses the presence

and location of regional nodal metastasis and the presence or absence of distant

metastasis. The most common sites of metastatic disease are the lung and bony

skeleton (Szklaruk et al., 2003).

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Table (4): Pathologic TNM staging system (Izumi et al., 1994)

Primary Tumor (T)

TX Primary tumor cannot be assessed histologically

T0 No evidence of primary tumor

T1 Solitary tumor without vascular invasion

T2 Solitary tumor with vascular invasion, or multiple tumors none more

than 5cm

T3 Multiple tumors more than 5cm or tumor involving a major branch

of the portal or hepatic vein(s)

T4 Tumors with direct invasion of adjacent organs other than the

gallbladder or with perforation of the visceral peritoneum

Regional Lymph nodes (N)

NX Regional lymph nodes cannot be assessed

N0 No regional lymph node metastasis

N1 Regional lymph node metastasis

Distant Metastasis (M)

MX Distant metastasis cannot be assessed

M0 No distant metastasis

M1 Distant metastasis

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Table (5): Stage grouping of TNM classification (Izumi et al., 1994).

Stage I T1 N0 M0

Stage II T2 N0 M0

Stage IIIA T3 N0 M0

IIIB T4 N0 M0

IIIC Any T N1 M0

Stage IV Any T Any N M1

The current pTNM system (Table 4) has been poorly predictive of tumor

recurrence after patients undergo hepatic resection or orthotopic liver

transplantation (OLTx) for HCC. This is borne out by the plethora of reports

citing a range of tumor and patient characteristics thought to be predictive of

tumor recurrence. Tumor free survival does not correlate well with TNM stage,

and recurrence outcomes within the stages are heterogeneous.(Izumi et al.,

1994).

The tumor node metastasis classification uses only tumor related

parameters (irrespective of the functional capacity of the liver) for identification

and follow up of HCC candidates for treatment. The TNM staging system,

although often used by surgeons for assessment of success of surgical resection

and liver transplantation, has been criticised for lack of prognostic value and has

been virtually abandoned. (Llovet et al., 1998).

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Okuda Staging System

Kunio Okuda suggested a staging system which provides a tool for

combined assessment of liver function and tumor load. It includes three stages

(Table 6) depending on tumor size (more or less than 50% of the liver area

affected) and the functional capacity of the liver, as assessed by albumin and

bilirubin levels and the presence of ascites. Positive criteria in the Okuda staging

system includes: Bilirubin >3g/dL, presence of ascites, albumin <30 g/dL and if

the tumor involves >50% of the liver. In Okuda stage I, no adverse parameter is

present. In Okuda stage II, one or two parameters are present and in Okuda stage

III, three or four parameters are present. Yet this new staging system still

requires some modifications as it lacks the means of assessment of vascular

invasion or "geographic tumor distribution within the liver lobes and is not

predictive enough for small tumors. (Okuda et al., 1985).

Table (6): Okuda classification (Shouval, 2002).

Four Criteria Tumor Size Ascitis Albumin Bilirubin

Cut off >50%

(+)

<50%

(-)

(+)

(-)

<30g/L

(+)

>30g/L

(-)

>3mg/dL

(+)

<3mg/dL

(-)

(-) (-) (-) (-)

1 or 2 (+)

Stage 1

Stage 2

Stage 3 3 or 4 (+)

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The Cancer of the Liver Italian Program (CLIP) Score:

This scoring system combines four variables (Table 7): The Child-Pugh

stage (A, B, or C) with tumor morphology (uni or multinodular with <50%

extension). AFP levels <400 or >400 IU/ml, and the presence or absence of

portal vein thrombosis as evidence of macro vascular invasion (CLIP

investigators, 2000).

Table (7): CLIP classification (CLIP investigators, 2003)

Variable Score 0 Score 1 Score 2

Child-Pugh class A B C

Tumor morphology Uninodular

<50% of liver volume

Multinodular

<50% of liver volume

Massive or

>50% of liver volume

AFP (IU/mL) <400 >400 ___

Portal vein thrombosis No Yes ___

Scores ranges from 0 to 6

AFP, α-fetoprotein; CLIP, Cancer of the Liver Italian Program

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The Barcelona – Clinic Liver Cancer Staging (BCLC) staging

This classification uses variables related to tumor stage, liver functional

status, physical status, and cancer-related symptoms, and links the four stages

described in TNM system with a treatment algorithm. In brief, patients at BCLC

stage 0 with very early HCC are optimal candidates for resection. Patients at

BCLC stage A with early HCC are candidates for radical therapies (resection,

liver transplantation or percutaneous treatments). Patients at BCLC stage B with

intermediate HCC may benefit from chemoembolization. Patients BCLC stage C

with advanced HCC may receive new agents in the setting of RCT, and patients

at BCLC stage D with end-stage disease will receive symptomatic treatment.

(Llovet, 2003).

It has been suggested that this classification is best suited for treatment

guidance, and particularly to select early stage patients who could benefit from

curative therapies. In that sense, it has recently been validated as the best staging

system in a cohort of patients with early HCC. (Cillo et al., 2004).

Several new systems have been proposed recently these include:

• The Chinese University Prognostic Index (CUPI).

• The Japan Integrated Staging (JIS).

• BALAD score.

The Chinese University Prognostic Index (CUPI):

CUPI score considers the following six predictive variables: TNM staging

system, total bilirubin, ascitis, alkaline phosphatase, alpha-fetoprotein and

asymptomatic disease on presentation. (Yeo et al., 2008).

Investigators in Hong Kong described in a prospective study in 726 HCC

patients scored with CUPI at disease presentation, the predictive power of CUPI

was compared with that of the TNM, Okuda staging systems and CLIP

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prognostic score. All patients were followed up for survival. Their results

estimated that CUPI score was more discriminant that the TNM and Okuda

staging systems or the CLIP prognostic score in classifying patients into

different risk groups and was better in predicting survival. (Yeo et al., 2008).

The Japan Integrated Staging (JIS) score:

Japan Integrated Staging (JIS) score accounts for both Child-Pugh

classification and tumor node metastasis (TNM) staging. The combined staging

system of hepatic function and tumor stage provides a better prediction of

prognosis (Nanshima et al., 2003).

The Japan Integrated Staging (JIS) sore has been reported to have good

stratification ability in patients with HCC. However, the JIS score could not

estimate malignant grade of HCC. Kitai et al., 2008 evaluated the performance

of a new staging system: the biomarker combined JIS (bm – JIS) which includes

3 tumor markers: Alpha-fetoprotien (AFP), Lens culinaris agglutinin – reactive

AFP (AFP-L3) and des--carboxy prothrombin (DCP) with the conventional JIS

score. The bm-JIS score showed superior stratification ability and thus was

found to be a better predictor of the prognosis than the conventional JIS score,

especially for the patients with good prognosis. (Kitai et al., 2008).

BALAD Score:

This new scoring system is based on 5 serum markers: bilirubin, albumin,

Lens culinaris agglutinin-reactive alpha fetoprotein (AFP-L3), alpha fetoprotein

(AFP), and des--carboxy prothrombin (DCP) and thus is termed the BALAD

score.

The new staging system for HCC combining serum albumin, serum

bilirubin, and 3 tumor markers predicts patient outcomes with excellent

discriminative ability. The system is easy to use and objective. In addition, stage

can be evaluated with the use of only 1 serum sample. It also allows global

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comparison of patients with HCC or comparison of patients from different time

periods with the same standard. (Hidenori et al., 2006). However, none of the

scoring systems is clearly superior to others, but the two most popular HCC

staging systems are the Okuda system and TNM classification of the

International Union Against Cancer (IUAC). (YU and Keefe, 2003).

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Serological Tumor Markers for HCC

An "ideal" tumor marker for HCC should be sensitive and specific

enabling to differentiate it at an early stage from premalignant lesions like

dysplastic nodules. In addition, the marker should be easily measurable,

reproducible and minimally invasive. (Yoon, 2008).

Serum tumor markers, as the effective method for detecting hepatocellular

carcinoma

• Oncofetal antigens and glycoprotein antigens.

• Enzymes and isoenzymes.

• Genes.

• Cytokines.

(Zhou et al., 2006).

Oncofetal Antigens and Glycoprotein Antigens:

Alpha fetoprotein (AFP) and alpha fetoprotein L3

Alpha fetoprotein (AFP) is a fetal specific glycoprotein synthesized from

fetal yolk sac, liver and intestines. Normally its serum concentration falls rapidly

after birth and its synthesis in adult life is repressed. However, greater than 70%

of HCC patients have a high serum concentrations of AFP because of tumor

excretion. Forty years after its discovery, serum AFP remains the most useful

marker for screening HCC patients. (Zhou et al., 2006).

Alpha fetoprotein is classified as a member of an albuminoid gene family,

which consists of four members: albumin (ALB), vitamin D-binding protein

(DBP), alpha fetoprotein (AFP), and alpha-Albumin (α ALB), termed afamin.

(Mizejewski, 2001). It is a glycoprotein consisting of 591 aminoacids that has

been reported to have single asparagine linked complex-type sugar chain.

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(Ferranti et al., 1995). The difference in the degree of fucosylation of the

asparagine linked sugar chain of AFP leads to heterogenecity of AFP. (Trojan et

al., 1989).

Total AFP can be divided into three different glycoforms, namely AFP –

L1, AFP – L2 and AFP – L3 according to their binding capability to lectin lens

culinaris agglutinin (LCA). AFP-L1, as the non – LCA – bound fraction, is the

major glycoform of AFP in the serum of non malignant hepatopathy patient. On

the contrary, AFP – L3, as the LCA bound fraction, is the major glycoform of

AFP in the serum of HCC patients. (Taketa et al., 2002).

Alpha fetoprotein may be falsely elevated due to benign liver diseases as:

neonatal hepatitis, viral hepatitis, fulminant toxic hepatitis and cirrhosis.

Microheterogeneity of the sugar component of AFP has been studied by affinity

chromatography and affinity electrophoresis with several lectins including:

concanavalin A (Con A) and lens culinaris agglutinin (LCA) having specificities

for different oligosaccarides. The Con A-reactive and LCA reactive species (L3)

of AFP was found in patients with HCC. (Brummund et al., 1980).

Serum AFP is the most widely used tumor marker in detecting patients

with HCC and has been proved to have capability of pre-figuring prognosis.

However, it has been indicated that AFP – L3 and DCP exel AFP in

differentiating HCC from non malignant hepatopathy and detecting small HCC.

(Zhou et al., 2006).

Screening for HCC has been attempted in high incidence areas such as

Africa, China, Taiwan, Japan, and Alaska. Initial large-scale screening in China

using less sensitive techniques (e.g. agglutinin and immunodiffusion, which

have cut-off values of 400 – 1000 IU/ml) was able to detect significant numbers

of new cases of this type of cancer. More sensitive methods (10 – 29 IU/ml)

using immunoassays, ultrasonography or both were employed in Taiwan and

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Japan with better success in detecting HCC at earlier stages. (Pascual et al.,

2008).

Alpha fetoprotein is useful for determining prognosis and monitoring of

therapy for HCC. The level of AFP is a prognostic indicator of survival.

Elevated AFP levels (>10 IU/ml) as well as serum bilirubin levels of greater

than 2 mg/dL are associated with shorter survival time. A significant increase of

AFP levels in patients considered free of metastatic tumor may indicate the

development of metastasis. It is a good indicator for monitoring therapy and

change in clinical status. Elevated AFP levels after surgery may indicate

incomplete removal of tumor or the presence of metastasis. Falling or rising

AFP levels after therapy may predict the success or the failure of the treatment

regimen. (Pascual et al, 1996).

Lens culinaris agglutinin – reactive Alpha fetoprotein (AFP-L3) isoform is

produced in HCC cells by attachment of an α 1,6 fucose residue on N-

acetylglucosamine of the carbohydrate chain on AFP. This carbohydrate

complex reacts with lens culinaris agglutinin (LCA). The AFP-L3 % is a ratio of

the LCA-reactive AFP to the total AFP. AFP-L3 is known to be a useful marker

for the diagnosis of HCC. Recent studies have shown that positive AFP-L3

results after treatment predicts tumor recurrence and poor clinical outcome.

(Carr et al., 2007).

In order to better understanding the role of lentil lectin-affinity AFP-L3 in

the diagnosis and differential diagnosis of HCC, Khein et al., applied the lectin-

affinity electrophoresis and antibody affinity blotting techniques to HCC

patients in Vietnam. Total AFP & AFP-L3% was measured in 65 patients with

histologically proven HCC and 25 patients with chronic liver disease (CLD). All

patients had serum AFP above 54 IU/ml AFP-L3% mean value in HCC patients

was 49.6 +/- 21.6%, which was significantly higher than that in the 25 CLD

patients (10.7 +/- 4.3%). When the cut-off level of AFP-L3% was set at 15%, the

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sensitivity was 96.9%, the specificity 92.0% and the accuracy 95.5% in the 65

HCC patients. They concluded that AFP-L3 is potentially a clinically useful

marker for the differentiation of increased AFP levels in HCC and CLD. The

AFP-L3 percentage is loosely related to HCC differentiation. (Khein et al.,

2001).

The serum concentration of 20 IU/ml is the most commonly used cut-off

value to differentiate HCC patients from healthy adults in clinical researches.

However, Soresi et al., 2003 evaluated the best cut-off value for AFP to

discriminate between liver cirrhosis (LC) and HCC in the studied group. 372

patients with LC and 197 patients with HCC were evaluated. Hepatic function

was estimated by the Child-Pugh's score and TNM classification for HCC

etiology was determined as viral or non viral. They found that the best cut-off

value for serum AFP in the studied population was 30 IU/ml, but at this level

sensitivity was low. This cut-off value was more useful in detecting non viral

HCC; therefore, there data confirm that the usefulness of AFP in the diagnosis

of HCC of viral etiology in limited, being more useful in HCC of non viral

etiology. The same results were concluded from a similar study by (Nguyen et

al., 2002).

HCC with a high AFP concentration (> 400 IU/ml) tend to have greater

tumor size, bilobar involvement, massive or diffuse types, portal vein

thrombosis and a lower median survival rate. (Li et al., 2005).

Although AFP has been used as a tumor marker for the diagnosis of HCC

for many years, its specificity is poor when levels fall within the "grey area" i.e.

10 – 500 IU/ml (Okuda, 1986). In this range, differentiation between benign and

malignant liver disease cannot be made with confidence on the basis of serum

AFP levels alone. There is thus a need to find a method of increasing the

specificity of AFP.

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Serum AFP can be determined by immunometric assay using either a

radioactive label or a enzyme label. Automated immunoassay systems to

measure AFP using these techniques are available. The detection limit of AFP

immunoassay is about 1 to 2 IU/ml. (Pascual et al., 1996).

Glypican – 3:

Glypican-3 (GPC3) is a heparin sulfate proteoglycan anchored to the

plasma membrane. It has been demonstrated to interact with growth factors and

modulate their activities (Capurro et al., 2003).

Expression alteration of the oncofetal protein Glypican-3 (GPC3) is

associated with several malignancies and has been identified as an

overexpressed gene in HCC. (Man et al., 2005).

Capurro et al., 2003 investigated whether GPC3 is overexpressed in liver

tissue sections in HCC – patients and whether GPC3 is detectable in the serum of

patients with HCC. GPC3 was assessed in liver tissue sections

immunohistochemisty and in serum by enzyme-linked immunosorbent assay.

Immunohistochemical studies showed that GPC3 was expressed in 72% of

HCCs (21 of 29), whereas it is not detectable in hepatocytes from normal liver

and benign liver diseases. Consistent with this, GPC3 was undetectable in the

serum of healthy donors and patients with hepatitis, but its levels were

significantly increased in 18 of 34 patients (53%) with HCC. They concluded

that GPC3 is specifically overexpressed in most HCCs and is elevated in the

serum of a large proportion of patients with HCC.

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Enzymes and Isoenzymes:

Gamma-glutamyl trasnferase

Serum gamma-glutamyl transferase (GGT) in healthy adults is mainly

secreted by hepatic Kupffer cell and endothelial cell of bile duct, and its activity

increases obviously in tissue of HCC and fetal liver. Total GGT can be divided

into 13 isoenzymes by using polyacrylamide gradient gel electrophoresis, and

some of them (I', II, II') can only be detected in the serum of HCC patients.

Sensitivities of GGT II have been reported to be 74.0% in detecting HCC and

43.8% in detecting small HCC. (Cui et al., 2003).

GGT is re-expressed during the development of HCC and can be divided

into several subfractions according to different electrophoresis mobility.

Hepatoma specific Gamma-glutamyl transferase HS-GGT is a part of total GGT

activity than can found only in sera of HCC patients so can be used as a specific

markr for HCC diagnosis (Yao et al., 1998).

Hepatoma specific GGT bands (including: I', II, and II', HS-GGT) is a

useful marker complementary to AFP for diagnosis of HCC. Combination of

both markers significantly increase the sensitivity over AFP alone. (Cui et al.,

2003).

Alpha – L – Fucosidase (AFU):

Alpha – L – Fucosidase (AFU) is a sort of enzyme to hydrolyze fucose

glycosidic linkages of glycoprotein and glycolipids.

Ishizuka et al., 1999 determined serum AFU activity in 73 patients with

liver cirrhosis (LC), the assay was repeated monthly for 42 months for

prediction of the development of hepatocellular carcinoma. HCC was diagnosed

in 27 patients (out of the studied 73 patients) by U/S during the observation

period. In 23 (85%) of the 27 patients diagnosed as HCC by sonography, serum

AFU activity was found to exceed 700 n mole/mL/h during the liver cirrhosis

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stage. In contrast, HCC developed in only 4 (9%) of 45 liver cirrhosis patients

with AFU activity below 700 n mol/mL/h. However, AFU activity was already

elevated in 23 (85%) of 27 patients at least 6 months before the detection of

HCC by sonography. So, the development of HCC can be predicted by means of

serial determinations of serum AFU activity in patients with liver cirrhosis.

Tangkijvanich et al., 1999 found that AFU is useful in early detection of

HCC, especially in conjunction with AFP and ultrasound, particularly in patients

with underlying viral hepatitis and cirrhosis.

Des – Gamma – Carboxyprothrombin:

Des – gamma – carboxyprothrombin (DCP), or prothrombin induced by

vitamin K absence or antagonist II (PIVKA – II), is an inactive prothrombin

deficient in gamma – carboxy – glutamic acid, which is produced by malignant

hepatocytes. DCP results from an acquired post translation defect in the

carboxylase system, independent of vitamin K deficiency (Weitz and Liberman,

1993).

A revised enzyme immunoassay EIA kit with increased sensitivity than

the conventional EIA has been development, while high specificity to HCC is

maintained (Okuda et al., 2000). A novel immunoassay using the

electrochemiluminescence (ECLIA) was developed to enable measurement of

low concentration of DCP (Shimizu et al., 2002).

Marrero et al., 2003 have conducted case control study to evaluate

whether DCP is more sensitive and specific than AFP for differentiating HCC

from non malignant liver disease in a group of American patients. G1, normal

healthy subjects, G2, patients with non-cirrhotic chronic hepatitis, G3, patients

with compensated cirrhosis and G4 patients with histologically proven HCC.

Assay of AFP and DCP were done. Both DCP and AFP levels increased

progressively from G1 to G4 but DCP values had less overlap among the groups

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than AFP. The determination of both DCP and AFP have been demonstrated to

be superior in monitoring HCC recurrence compared with each marker alone.

However, elevated DCP or serum AFP levels are both predictors of poor

prognosis. Based on these findings, DCP is considered to be an independent

marker for detection and follow-up of HCC, but it is not sensitive for early

diagnosis of HCC. (Inoue et al., 1994).

GENES

Alpha-fetoprotein mRNA:

HCC cells spread into blood circulation and become the source of

recurrence after operation. This may be the primary reason for the unsatisfactory

long-term survival after surgery.

The presence of circulating HCC cells may be indicative of metastasis if

AFP mRNA is detected in peripheral blood (Chen et al., 2002).

Ijichi et al., 2002 have indicated that serum AFP mRNA detected by

reverse-transcription polymerase chain reaction (RT-PCR) may be a valuable

indicator of poor prognosis for HCC patients.

The expression of AFP mRNA is correlated with portal thrombosis,

nodules of tumor, tumor diameter, and TNM stage. The recurrence-free interval

of HCC patients with postoperative serum AFP mRNA positively has been

reported to be significantly shorter than that of HCC patients with post operative

negativity.

Gamma-glutamyl transferase mRNA

GGT mRNA can be detected in the serum and liver tissues of healthy

adults or patients with HCC, nonmalignant hepatopathy, hepatic benign tumor,

and secondary carcinoma of liver. It can be divided into three types: fetal liver

(type A), HepG2 cells (type B), and placenta (type C). Type A is predominant in

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normal liver tissues or liver tissues with nonmalignant hepatopathy, benign

tumor, and secondary carcinoma. On the contrary, type B is predominant in

cancerous tissues of HCC. During the development of HCC, the expression of

GGT mRNA in liver tissues may shift from type A to type B. (Tsutsumi et al.,

1996).

It has been indicated that HCC patients with positive type B would have a

worse outcome, earlier recurrence, and more post-recurrence death. (Sheen et

al., 2003).

The expression of tissues type B may be a valuable indicator of poor

prognosis in HCC. Serum levels of type B have also been reported to be

significantly higher in HCC patients than in healthy adults. Therefore, serum

type B may be an available supplementary to AFP in the diagnosis of HCC.

(Han et al., 2003).

Human telomerase reverse transcriptase mRNA

Telomerase reverse transcriptase (hTERT) mRNA has been reported to

detectable in the serum of patients with breast cancer. Furthermore, it has also

been demonstrated to be a novel and available marker for HCC diagnosis. The

expression of hTERT mRNA in the serum of HCC patients is significantly

higher than that in the serum of healthy adults or patients with nonmalignant

hepatopathy. (Miura et al., 2005).

The use of newly developed real-time quantitative reverse transcription

polymerase chain reaction may improve the effectiveness of determination. It

has been reported that the sensitivity and specificity of hTERT mRNA in

detecting HCC are 88.25% and 70%, respectively, which excel those of

conventional tumor marker, such as AFP mRNA, AFP and DCP. Moreover, it

has been indicated that the expression of serum hTERT mRNA, may be a

valuable indicator of poor prognosis for HCC patients. (Miura et al., 2003).

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CYTOKINES

Vascular endothelial growth factor

Vascular endothelial growth factor (VEGF) is a secreted cytokine

that positively regulates tumor neovascularization.

Zachary, 2003 have suggested that angiogenesis is essential in tumor

growth and progression, including that of HCC, which are typically

characterized by a high level of vascularization. In fact, it has been shown that

the expressions of VEGF in cancerous tissues of HCC and HCC with

microscopic venous invasion are significantly higher than that in normal liver

tissues and HCC without microscopic venous invasion. HCC patient with over-

expression of VEGF have a lower survival rate. (Huang et al., 2005).

Platelets have been reported by kim et al., 2004 to act as transporters of

tumor-originated VEGF. It has been indicated that serum VEGF per platelet

count, as an indirect theoretical estimate of VEGF in platelets, in HCC patients

is significantly higher than that in healthy adults and patients with nonmalignant

heptopathy (and the high serum VEGF per platelet count) is associated with

advanced stage of HCC, portal vein thrombosis, poor response to treatment, and

shorter overall survival, Therefore it may be an available diagnostic or

prognostic indicator for HCC.

Interleukin – 8

Interleukin-8 (IL-8) is a multifunctional chemokine that affects human

neutrophil functions, including chemotaxis, enzyme release, and expression of

surface adhesion molecules. It has direct effects on tumor and vascular

endothelial cell proliferation, angiogenesis, and tumor migration. Recent

researches have indicated that IL-8 regulates tumor cell growth and metastasis in

liver (Akiba et al., 2001).

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Ren et al., 2003 reported that the preoperative serum IL-8 levels in HCC

patients are significantly elevated compared with those in healthy adults and its

high serum levels correlate with a large tumor size (>5cm), absence of tumor

capsule, presence of venous invasion, advanced pathological tumor-node-

metastasis stage, and a poor disease-free survival. Therefore, it may be an

available diagnostic or prognostic indicator for HCC.

Tumor specific growth factor

Malignant tumor can release tumor-specific growth factor (TSGF), which

results in blood capillary amplification surrounding the tumor, into peripheral

blood during its growing period. Therefore, the serum levels of TSGF can reflect

the existence of tumor. It has been indicated that TSGF can be used as a

diagnostic marker in detecting HCC, and its sensitivity can reach 82% at the cut-

off value of 62 U/mL. Furthermore, the simultaneous determination of TSGF

and other tumor markers has been shown to give a higher accuracy. (Zhu et al.,

2004).

The simultaneous determinations of TSGF (at the cut-off value of 65

U/mL), AFP (at the cut-off value of 25 ng/ml) and serum ferritin (at the cut-off

value of 240 µg/mL) have a sensitivity of 98.4% and specificity of 99%. (Pan et

al., 2004).

There are some other markers, which could be used as diagnostic or

prognostic indicators for HCC, in this category. It has been reported that the

determination of serum insulin-like growth factor-II (IGF-II) (at the cut-off

value of 4.1 mg/g, prealbumin) has a sensitivity of 63%, specificity of 90%, and

accuracy of 70% in the diagnosis of small HCC. Moreover, the simultaneous

determination of IGF-II and AFP (at the cut-off value of 50 ng/mL) can improve

the sensitivity to 80% and accuracy to 88%. The over-expression of granulin-

epithelin precursor (GEP) in cancerous tissues of HCC is associated with venous

infiltration and early intrahepatic recurrence. (Tsai et al., 2005).

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3 – Squamous Cell Carcinoma Antigen

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Squamous Cell Carcinoma Antigen Squamous Cell Carcinoma Antigen (SCCA) belongs to the high

molecular weight family of Serine Proteinase Inhibitors (Serpins) (Suminami et

al., 1991). Over 1000 serpins have now been identified, these include 36 human

proteins, as well as molecules in plants, fungi, bacteria and certain viruses.

(Hunt and Dayhoff, 1980). In mammals, inhibitory-type serpins regulate serine

proteinasis involved in, for example, coagulation, fibrinolysis, inflammation,

cell migration and extracellular matrix remodeling (Potempa et al., 1994).

It was originally isolated by Kato and co-workers from human squamous

cell carcinoma tissue and shown to consist of at least 10 subfractions differing in

isoelectric point (Kato and Torigoe, 1977).

SCCA is a tumor-associated protein of squamous cell carcinoma of

various organs (Suminami et al., 1998). Biochemical analysis of SCCA by

sodium dedecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) reveals

a single band with a molecular mass of ~ 45 KDa (Kato et al., 1984).

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Seprins

Serpins are members of a family of structurally related protein inhibitors

of serine proteinases, with molecular masses between 40 and 100 K Dalton

(Gettins et al., 2005). However, serpins that inhibit caspases (Ray et al, 1992)

and papain like cysteine proteases (Schick et al., 1998) have also been

identified. Rarely, serpins perform a non-inhibitory function; for example,

several human serpins function as hormone transporters (Pemberton et al.,

1996) and certain serpins function as molecular chaperones (Nagata, 1996) or

tumor suppressors (Zou et al., 1994).

Inhibitory serpins

Inhibitory serpins are relatively large molecules about 330 – 500 amino

acids. They are "suicide" or "single use" inhibitors that use a unique and

extensive conformational change to inhibit proteases (Huntington et al., 2000).

The major conformational change that occurs within both the protease and the

serpin as a result of serpin-enzyme complex formation provides an elegant

mechanism for cells to specifically detect and clear inactivated serpin-protease

complexes. This conformational mobility renders serpins heat labile and

vulnerable to mutations that promote misfolding, spontaneous conformational

change, formation of inactive serpin polymers and serpin deficiency (Carrell

and Lomas, 1997).

In humans, several conformational disease or serpinopathies linked to

serpin polymerization have been identified, including emphysema (antitrypsin

deficiency) (Lomas et al., 1992) thrombosis (antithrombin deficiency) and

angioedema (C1 esterase inhibitor deficiency) (Aulak et al., 1998).

Accumulation of serpin polymers in the endoplasmic reticulum of serpin-

secreting cells can also result in disease most notably cirrhosis and familial

dementia. Other serpin-related diseases are caused by null mutations or (rarely)

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point mutations that alter inhibitory specificity or inhibitory function (Stein and

Carrell, 1995).

Broad organization of the serpins:

The two largest clades of the 36 human serpins that have been identified

are: clade A serpins and clade B serpins. The extracellular "clade A" molecules

includes thirteen members (SERPIN A1 – A13) and are found on chromosomes 1,

14 and X. Clade A serpins include inflammatory molecules such as SERPIN A1

(antitrypsin) and SERPIN A3 (antichemotrypsin) as well as non-inhibitory

hormone transport molecules as SERPIN A6 (corticosteroid binding globulin)

and SERPIN A7 (thyroxine-binding globulin) (Law et al., 2005).

The intracellular "clade B" serpins include thirteen members (SERPIN B1

– B13) and are found on chromosomes 18 and 6. Clade B serpins include

inhibitory molecules that function to prevent inappropriate activity of cytotoxic

apoptotic proteases (SERPIN B6 , also called P16) and inhibit papain-like

enzymes (SERPIN B3 , also called squamous cell carcinoma antigen-1).

However, SERPIN B4 inhibits cathepsin G and chymase also called: (squamous

cell carcinoma antigen-2). Non inhibitory serpin (SERPIN B5) also called:

maspin, functions to prevent metastasis in cancer breast and other cancers

through an incompletely characterized mechanism (Silverman et al., 2001).

Other human serpins include: Antithrombin (SERPIN C1), plasminogen

activator inhibitor 1 (SERPIN E1) C1 inhibitor (SERPIN G1) and others. In

humans, the majority (27 out of 36) of serpins are inhibitory (Law et al., 2006).

Structural biology of the serpins

Serpins are made up of three Beta sheets (A, B, C) and 8 – 9 alpha

helices. The region responsible for interaction with target proteases, the reactive

center loop (RCL), forms an extended, exposed conformation above the body of

the serpin scaffold. Following proteolysis, the amino-terminal portion of RCL

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inserts into the center of the β–sheet A to form an additional (fourth) strand.

This conformational transition is termed: the stressed (S) to relaxed (R)

transition, as the cleavage of native inhibitory serpin results in a dramatic

increase in thermal stability. Serpins use the S – to – R transition to inhibit target

proteases (Huntington et al., 2000).

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Genetics and Isoforms of Squamous cell carcinoma antigen

Recent molecular studies show that the SCCA is transcribed by two

almost identical genes: SCCA1 and SCCA2 (Schneider et al., 1995) both of

them have been located on chromosome 18q21.3 (Murakami et al., 2000). The

more telomeric gene, was designated SCCA1, whereas the more centromeric

gene was designated SCCA2.

SCCA1 and SCCA2 encode for proteins that are 98% and 92%

homologous at the nucleotide and amino acid levels, respectively (Cataltepe et

al., 2000). Although the two forms are encoded by two separate genes,

alternative splicing or post translational modifications such as N-glycosylation

could account for the two forms of the protein SCCA1 and SCCA2 (Schneider et

al., 1995).

On the basis of the deduced amino acid sequences, SCCA1 corresponds to

the neutral (PI 6.4) and SCCA2 the acidic (PI 5.9) isoform of the original SCCA

complex (Cataltepe et al., 2000).

Although SCCA1 (SERPIN B3) and SCCA2 (SERPIN B4) are almost

identical members of the seprin family, significant differences in their reactive

site loops (that part of the molecule that is bound by the active site of the

proteinase) suggest that they inhibit different classes of proteinases (Cataltepe et

al., 2000).

Biochemical analysis shows that SCCA1 is a cross-class inhibitor of

papain-like cysteine proteinases, such as cathepsins L,S and K whereas SCCA2

inhibits chemotrypsin-like serine proteinases, cathepsin G, and mast cell

chymase (Schick et al., 1997). These findings may have important diagnostic

and therapeutic implications, because the balance between proteinases and

inhibitors can affect tumor cell motility, invasiveness, proliferation, and death

(Cataltepe et al., 2000).

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Expression pattern of SCCA isoforms:

The neutral isoform of SCCA (SCCA1) is detected in both malignant and

normal epithelial cells (Mueller and World, 1989). In contrast, the acidic form

SCCA2 is present in the tumor cells, especially those located at the periphery of

the tumor, and in the sera of cancer patients with well differentiated SCC (Kato

et al., 1987).

SCCA is not specific for malignant tissues, however, as the protein is

detected in the suprabasal levels of normal stratified squamous epithelia of the

skin and mucous membranes and in the pseudostratified ciliated columnar

epithelia of the conducting airways (Kato et al., 1987).

SCCA1 and SCCA2 proteins co-localized to the skin, esophagus, tonsils,

tongue, thymus, trachea, bronchi, vagina, and uterine cervix. This co-

localization pattern was also observed in most cases of head and neck Squamous

Cell Carcinomas (SCCs) and lung SCCs (Cataltepe et al., 2000).

In normal adult skin, Cataltepe et al., 2000 detected weak and focal

SCCA1 and SCCA2 immunoreactivity only in the outer root sheath of selected

vellus and terminal hair follicles and in some of these cases, in the epidermis

immediately adjacent to these structures. SCCA immunoreactivity in normal

squamous epithelium of the skin was reported in several studies (Mino-

Miyagawa et al., 1990 ; Horiuchi et al., 1994). By radio-immunoassay, Mino-

Miyagawa et al., 1990 showed that SCCA concentration in normal skin

epithelium is comperable to that in the squamous epithelia of vagina, cervix,

esophagus and SCCs of the cervix. In contrast to these reports, Morioka (1980)

and Duk et al., (1989) found either low or absent SCCA expression in the skin.

Cataltepe et al., 2000 showed that both SCCA1 and SCCA2 were present

in the stratified squamous epithelium of the digestive system lining the mucosal

surfaces of the tongue, tonsils, and esophagus, but they did not detect SCCA1 or

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SCCA2 immunoreactivity in submandibular, parotid, or minor salivary glands.

Their results suggest that SCCA1 and SCCA2 in saliva were derived from the

squamous epithelial cells lining mucosal surfaces of the upper digestive tract

rather than from the salivary glands.

Mino-Miyagawa et al., 1990 reported the presence of SCCA in areas of

squamous metaplasia within the lung. Therefore, it appears that SCCA1 and

SCCA2 may be co-expressed in non-squamous epithelia undergoing squamous

metaplastic changes in response to inflammation or infection.

SCCA1 and SCCA2 expression was not restricted to squamous epithelial

cells. SCCA1 and SCCA2 were detected in the pseudo-stratified columnar

epithelium of the conducting airways. Diseases such as asthma, cystic fibrosis,

and emphysema are characterized by an influx of inflammatory cells and the

release of proteinases that degrade the structural components of the lung.

Because SCCA1 and SCCA2 are inhibitors of serine and cysteine proteinasis,

their location in the bronchial mucosa would place them in an ideal position to

protect the airways from proteinases derived from inflammatory cells, epithelial

cells, and microorganisms (Cataltepe et al., 2000).

In the female genitourinary system, cataltepe et al., 2000 detected

comperable SCCA1 and SCCA2 staining in the supra-basal layers of the

stratified squamous epithelium of the uterine cervix and vagina. Kato et al.,

1987 reported that a monoclonal antibody that binds both the acidic and neutral

isoforms of SCCA stained the intermediate layers of the normal squamous

epithelium of the cervix.

In head, neck, and lung squamous cell carcinomas SCCs, Cataltepe et al.,

2000 detected SCCA1 and SCCA2 immunoreactivity in well-differentiated

squamous cell components of tumor tissues. This pattern of expression was

similar to that observed in similar studies using non discriminatory antibodies

raised against the SCCA (Kearsley et al., 1990).

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Role of SCCA in diagnosis of different diseases

SCCA is a serological marker of squamous cell carcinomas of the uterine

cervix, vulva, lung, head & neck, and esophagus (De Bruijin et al., 1998),

(Vassiliakopoulos et al., 2001) and (Snyderman et al., 1995).

Although SCCA1 and SCCA2 are detected in the cytoplasm of normal

squamous epithelial cells, neither serpin is detected normally in the serum. Thus

their presence in the circulation at relatively high concentrations suggests that

malignant epithelial cells are re-directing serpin activity to the fluid phase via an

active secretory process. A change in the distribution pattern of SCCA1 and

SCCA2 (i.e. intracellular to extracellular) could indicate the need of tumor cells

to neutralize harmful extracellular proteinases (Uemura et al., 2000).

SCCA levels in cancer cervix:

The delineation of tumor spread and the early detection of tumor

recurrence and/or progression are the major problems in the management of

patients with squamous cell carcinoma of the cervix (Neunteufel et al., 1989)

SSCA is a serological marker of squamous cell carcinomas. SCCA isoforms

were determined by Roijer et al., 2006 before therapy and in follow up samples

from patients with cervical cancer and in serum samples from healthy females.

Rising levels of SCCA1 and SCCA2 were seen in patients with recurrence or

progressive disease at the end of the study. The rise in SCCA2 was usually more

prominent than that in SCCA1. Both SCCA1 and SCCA2 were elevated in serum

from cervical cancer patients and followed the clinical course of the disease

during therapy monitoring.

In squamous cell carcinoma of the uterine cervix, pretreatment serum

SCCA may be used as an early stage prognostic factor (Duk et al., 1997) and the

use of pretreatment SCCA has been suggested in order to select high-risk

patients for adjuvant therapy (De Bruijin et al., 1998).

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Gaarenstroon et al., 2000 reported that, SCCA is a relatively new, non

invasive tumor marker in cancer cervix. It helps not only in monitoring response

to the therapy but also in detecting its early recurrence. This study is in

agreement with the previous studies showing a correlation between SCCA and

stage, as the plasma concentration depends on the tumor load, and SCCA values

increased with advancing stages. The mean values of SCCA in all stages (I-IV)

were above the cut-off normal values of 2.5µg/mL. SCCA is a reflection of

tumor bulk, intrinsic biologic tumor characteristics, and invasiveness of tumor

(Juang et al., 2000).

De Bruijin et al., 1998 reported a decrease of SCCA in 91% of cases who

received chemotherapy before radiotherapy. A positive and significant

correlation was seen between SCCA and tumor volume post-chemotherapy. Pre-

chemotherapy SCCA also predicts response to chemotherapy and when SCCA

was between 5 and 30 µg/mL, a significant response to chemotherapy was seen.

A similar response was seen by Ohno et al., 2003 when SCCA was studied in

relation to radiation.

SCCA levels in lung cancer

With regard to lung cancer, some markers have a good sensitivity and

specificity in the small cell subtype (SCLC) like the neuron-specific enolase or

BB creatine kinase. However, with regard to non-small cell lung cancer

(NSCLC) subtypes, the well-known markers basically carcinoembryonic antigen

(CEA) are not sufficiently sensitive or specific (Jaques et al., 1988).

DeCos et al., 1994 have carried out several studies oriented to the clinical

application of SCC-Ag in lung cancer (LC). In these studies, the sensitivity of

SCCA in LC is not high (27.6%). Other authors have found figures of

sensitivity that vary from 33% (Mizushima et al., 1990) to 78.4% (Ebert et al.,

1990). The differences depend on the analytical method and the normal range

adopted. In the study of Body et al., 1990, the sensitivity was 35% for the

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squamous LC and clearly lower for the other types. DeCos et al., 1994 found the

highest sensitivity for the SCC type (47.7%), with an important difference over

the other types (14.3% for adenocarcinoma, 9.2% for SCLC and 6.7% for large

cell LC). Concerning the diagnostic value, the increased serum levels of this

marker seem fairly specific for malignant disease. Body et al., 1990 found

elevated values of SCCA in 11.1 percent of 90 patients with benign pulmonary

disease. This specificity (88.9%) was equal to that of CEA.

DeCos et al., 1994 found a significant association between the initial

serum SCCA and the survival time. This association is not surprising since the

marker has a good correlation with the tumor burden and stage. These results are

just like other authors as Body et al., 1990.

SCCA in cancer larynx

Lachowicz et al., 1999 studied the clinical usefulness of SCCA in patients

with squamous cell carcinoma of the larynx. Plasma specimens were obtained

from 70 patients with cancer of the larynx before and after treatment and during

follow-up. Disease status and the marker levels were determined blind to each

other. Microparticle enzyme immunoassay was used to measure the SCC-Ag

level. Applying standard normal limits, the sensitivity of the marker at diagnosis

was 25.7%. SCC-Ag levels were generally lower after therapy than before.

Relapse occurred more often in patients with an abnormal pretreatment SCC-Ag

level, which was more frequent in those with nodal invasion. The marker level

increased in 70% of the patients with relapse before the clinical detection of

recurrence. SCC-Ag is of limited usefulness in the primary diagnosis of cancer

of the larynx, but is useful in detecting recurrence of cancer.

SCCA in head and neck cancer

Patients with head and neck tumors (HNT) have a high risk of early

locoregional relapse that is difficult to diagnose. Banal et al., 2001 evaluated the

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usefulness of SCCA for monitoring such patients compared with CYFRA 21-1

assay. 312 HNT patients, including 204 newly diagnosed patients, were

followed up for a median of 446 days with serial serum assays for SCC and

cyfra 21-1. Untreated patients showed SCC and CYFRA 21.1 serum levels

correlated with each other. Concentrations were correlated to clinical stage,

tumor size and nodal status. CYFRA 21-1, but not SCC was related to the

presence of metastasis and the primary tumor size.

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SCCA in hepatocellular carcinoma

Pontisso et al., 2004 evaluated SCCA by immunohistochemistry in 65

HCC patients of different etiology and in 20 normal subjects. Squamous cell

carcinoma antigen was detected in 55 out of 65 (85%) tumor specimens, but in

none of the 20 controls. In the majority of the cases, squamous cell carcinoma

mRNA could be directly sequenced in 14 out of 18 liver tumors but in none of

the corresponding non tumor samples.

Giannelli et al., 2005 investigated 120 patients with HCC and 90 patients

with liver cirrhosis. Both serologic markers for HCC were measured: squamous

cell carcinoma antigen (SCCA) and alpha fetoprotein (AFP).

The authors reported that as a marker of HCC, SCCA has high sensitivity

(84.2%) but low specificity (48.9%). However, the combination of AFP and

SCCA yielded a correct serologic diagnosis in 90.83% of HCC patients. A small

percentage of patients remained undetected, likely because of the low specificity

of SCCA. They concluded that the combined use of AFP and SCCA represents a

more powerful tool for the serologic detection of HCC.

In another study by Giannelli et al., 2005 the expression of SCCA was

investigated in tumoral and peritumoral tissues and in the serum of 52 HCC

patients, as well as in the serum of 48 cirrhotic patients. The results showed that

SCCA expression was much stronger in the tumoral than in the peritumoral

tissue of HCC. Moreover, it is also evident in metastatic nodules present in the

peritumoral tissue. SCCA serum levels were significantly higher in HCC

samples than in cirrhotic samples. However, no correlation was found between

SCCA expression and the HCC histologic degree, nor did SCCA expression

correlate with tumor size, presence of metastasis or clinical outcome. They

concluded that in HCC patients, the SCCA antigen could represent a useful

marker for the detection of micro-metastasis in the tissues and for large-scale

screening of serum in patients at risk.

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Beneduce et al., 2005 assessed the presence of squamous cell carcinoma

antigen (SCCA), as a free form and complexed with immunoglobulins, in serum

from 50 patients with HCC, 50 patients with cirrhosis, 50 patients with chronic

hepatitis and 73 healthy controls. The authors compared SCCA measurement

with the measurement of alpha fetoprotein (AFP) levels. They found that

circulating immune complexes (ICs) composed by SCCA and immunoglobulin

M (IgM) IC (SCCA – IgM IC) were undetectable in serum from a healthy

control population (0 of 73 controls); however, 35 of 50 patients with HCC

(70%) were reactive for SCCA- IgM IC independent of etiology. Among

cirrhotic patients, SCCA – IgM IC was elevated in 13 of 50 patients (26%) but

at lower levels than HCC, whereas 9 of 50 patients with chronic hepatitis (18%)

were reactive. However, AFP levels were elevated significantly in only 21 of 50

patients with HCC (42%). By using an AFP cut-off value of 20 IU/ml, 96% of

patients with HCC were positive for at least one marker (either AFP or SCCA –

IgM IC).

The study results indicated that SCCA – IgM IC represents novel

serologic biomarkers, which, alone or in combination with AFP, can increase the

sensitivity for diagnosing HCC significantly.

Giannelli et al., 2007 studied in a large number of patients, the diagnostic

accuracy of combined serological biomarkers (AFP, AFPIC, SCCA and

SCCAIC) for HCC detection. The studied group included 961 patients. Inclusion

criteria were: age over 18 years and the presence of HCC or liver cirrhosis and

exclusion criteria were other concomitant cancers. Among the patients enrolled,

499 were classified as HCC based on U/S, CT and liver biopsy. 462 patients

with liver cirrhosis diagnosed by clinical and biochemical parameters. Serum

samples were collected and analyzed by ELISA kit for AFP, AFPIC, SCCA and

SCCAIC.

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The authors detected that SCCA was inversely correlated with tumor size,

while AFPIC and SCCAIC were not. These findings suggest that the use of

these markers may help in the detection of early HCC onset.

The absence of correlation between AFP and the other markers

investigated suggests that each marker is related to a different aspect in HCC so

that use of them in combination could significantly improve the diagnostic

accuracy. In fact, the use of additional biomarkers such as AFPIC, SCCA,

SCCAIC allowed them to identify a new group of HCC patients, even in the

absence of increased AFP levels. In short, the efficiency of HCC detection was

significantly improved by the use of all these markers together, although as

expected this increased the number of false positives.

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Subjects and Methods


Subjects:

The present study was conducted on 85 subjects. They were

divided into two groups:

I- Patients' group:

This group comprised 65 newly diagnosed patients with hepatic

focal lesion accompanied with liver cirrhosis from those attending the

Tropical Medicine Department – Cairo University from May 2006 till

March 2007.(Group I)

Group I was further subdivided according to their histopathologic

findings into:

GROUP Ia: it included 49 patients with proved hepatocellular

carcinoma on top of cirrhotic liver.

GROUP Ib: it included 16 patients with hepatic cirrhosis only.

II- Control group:

This group comprised 20 healthy age and sex matched individuals.

They were free as proved by their clinical examination as well as liver

function tests.

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All subjects underwent the following:

1- Clinical history and physical examination:

History:

A complete history was taken including:

Age, sex, occupation, affection with schistosomiasis, duration of

liver disease, abdominal pain, jaundice, abdominal distension, edema of

lower limbs, haematemesis, melena, encephalopathy and coma.

Examination:

General and local examination were done for the presence of

jaundice, encephalopathy, foetor hepaticus, spider nevi, edema of lower

limbs, flapping tremors. The liver was examined for size, consistency,

surface and tenderness. The spleen size and the presence or absence of

ascitis were noted. Each patient was assigned a grade using risk grade of

Child-Pugh score.

2- Abdominal Ultrasonography:

It was done for all subjects using convex probe at 3.5 Mhz Toshiba

(ECCOCCE) SSA – 340A to document the presence of hepatic focal

lesions, number, size, site and pattern of focal lesion were commented

upon if present. Cirrhosis was diagnosed in the presence of irregular

surface, coarse texture and attenuated hepatic veins. (Edmondson and

Steiner, 1954). (Figure 5).

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Figure (5): Ultrasound Picture: Hyperechoic focal lesion (HCC)

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Only patients with hepatic focal lesion underwent

the following:

A- Triphasic Abdominal CT

Patients with a focal lesion on US were further investigated with

CT scan. The typical specific enhancement pattern for the diagnosis of

HCC is the arterial uptake followed by venous washout in the delayed

portal/venous phase. Tumor size was defined as the maximum diameter

of tumor nodes measured at the time of assessment. (Figure 6).

Figure (6): Arterial phase of spiral CT: Full enhancement of HCC with

feeding vessel (arrow).

B- Liver biopsy

1- Preparation of the patients

• Prothrombin time was done and if it was more than 3 seconds

prolonged over the control value, the patient was given 10mg vit K

intramuscular for 3 days, after which prothrombin time was

repeated.

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• In cases where ascitis was present, measures were taken to

minimize it.

2- Biopsy procedure

For non-enhancing lesions, histopathology was made using US

guided fine needle biopsy (a 18 – gauge Tru Cut needle). All biopsies

results were graded by Steiner – Edmondson grading system (Michael et

al., 2001). Patients with cholangiocarcinoma, hepatoblastoma and liver

metastasis were excluded. (figure 7).

Figure (7): Histopathology of HCC (grade II).

Laboratory Tests for all subjects

Sample collection and storage

Five ml venous blood were collected from all subjects in blank

vaccutainers, they were left to clot, centrifuged at 1000 rpm, then sera

were divided into aliquots. They were stored at -70°c till the assay date of

SCC-Ag. Sera for liver functions and AFP were immediately assayed.

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Two ml were collected for assay of prothrombin time, nine

volumes of blood were added to one volume of 31.3 g/L aquous

trisodium citrate dehydrate. Plasma samples were separated by

centrifugation at 2000 rpm for 15 min. Plasma was kept at room

temperature for testing within two hours as the preferred schedule.

A- Routine laboratory investigations:

- Serum AST, ALT, bilirubin and albumin were done on Hitachi 917

instrument by routine analytical methods (Roche Diagnostics GmbH, D.

68298 Mannheim).

- Prothrombin time was done by coagulometers Hospitex single channel

coagulometer (Hospitex Diagnostics via S. Piero a Quaracchi, 224 –

50145 FIRENZE – ITALY).

- Hepatitis markers by Microparticle Enzyme Immunoassay (MEIA)

(Engvall and Perlmann, 1971) using AxSym autoanalyser (ABBOTT

LABORATORIES Diagnostic Division Max – Planck – Ring 65205

Germany).

B- Special laboratory tests including Alpha Fetoprotein and

Squamous cell carcinoma antigen determination:

Determination of Alpha Fetoprotein (AFP):

Sera from selected patients and controls were used for estimation

of serum level of AFP by immunoradiometric assay (IRMA) using Coat –

A – Count AFP IRMA kit provided by DPC (Diagnostic products

corporation, 157700 west 96m street Los Angeles, CA90045 – 5597)

(Dudley et al., 1985).

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Principle of the Method:

Coat-Count AFP IRMA is a solid phase immunoradiometric assay.

The assay utilizes 125I-labeled anti AFP polyclonal Ab in liquid phase,

and monoclonal anti-AFP Ab immobilized to the wall of a polystyrene

tube. In the procedure:

• AFP binds to the monoclonal Ab coated to the tube.

• Polyclonal tracer binds to the immobilization AFP/anti AFP

complex.

• Unbound tracer is removed by decanting and washing the tubes.

• The tube is counted In a gamma counter for one minute. The

concentration of AFP in the patient sample is directly proportional

to the number of counts per minute.

The AFP concentration is determined by comparing the number of

counts with those obtained from the set of calibrators provided.

Immunometric Assay Procedure:

All components were brought to room temperature (15 – 28°)

before use.

• 14 AFP Ab coated tubes were labeled A (non-specific binding) and

B through G (maximum binding) in duplicate. Additional AFP Ab

coated tubes were labeled for controls and patients samples.

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Calibrator IU/ml

A (NSB) 0

B 2.0

C 10

D 25

E 50

F 150

G (M.B) 300

• 25 µL of each calibrator, control and patients serum samples were

pipitted directly to the bottom into the tubes prepared. Patients

samples expected to contain high concentration were suitably

diluted with the zero calibrator before assay. A disposable-tip

micropipette was used. Changing the tip between samples was

done to avoid errors due to carry over.

• 200 µL of 125I AFP Ab was added directly to the bottom of every

tube. Sample and tracer were then thoroughly mixed.

• The tubes were shaken for 60 minutes on a rack shaker.

• Then the tubes were decanted thoroughly. 2.0ml of buffered wash

solution were added to each tube. The tubes were decanted

thoroughly after 1 – 2 minutes. Then this step was repeated again.

• Removing all visible moisture would greatly enhance precision.

After the second wash, all the tubes were left to drain for 2 or 3

minutes. Then the tubes were stricken sharply on absorbent paper

to shake off all residual droplets.

• Then the tubes were counted for 1 minute in a gamma counter.

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Calculations:

To calculate results (in terms of concentration units) from a log-log

representation of the calibration curve, first the counts per minutes (CPM)

were corrected by subtracting the average CPM of the non-specific

binding tubes (calibrator A):

Net counts = Average count – Average NSB counts.

Then the percent binding was determined (relative to that of the

highest calibrator) here called "%B/MB" as a percent of "maximum

binding".

With the NSB – corrected counts of the highest calibrator taken as

100%.

Percent bound = (Net counts / Net MB counts) x 100. Using 3

cycle log-log graph paper, percent bound was plotted versus

concentration for each of the non zero calibrators and a curve was drawn

approximating the path of these points. Concentrations for controls and

unknowns within the non-zero calibrators were estimated from the

calibrator curve by interpolation.

Determination of Squamous Cell Carcinoma Antigen (SCC-Ag)

Sera from selected patients and controls were used for estimation

of SCC-Ag using CanAg SCC EIA provided by CanAg Diagnostics AB,

SE – 414 55 Gothenburg, Sweden (Herberman., 1979).

Principle of the test

The CanAg SCC EIA is a solid phase, non-competitive

immunoassay based upon the direct sandwich technique. Calibrators and

patient samples are incubated together with biotinylated Anti-SCC

monoclonal antibody in Streptavidin coated microstrips. After washing

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buffered Substrate/Chromogen reagent (hydrogen peroxide and 3, 3', 5, 5'

tetra-methylbenzidine) is added to each well and the enzyme reaction is

allowed to proceed. During the enzyme reaction a blue colour will

develop if antigen is present. The intensity of the colour is proportional to

the amount of SCC present in the samples.

The colour intensity is determined in a microplate

spectrophotometer at 620 nm (or optionally at 405 nm after addition of

Stop Solution). Calibration curves are constructed for each calibrator. The

SCC concentrations of patient samples are the read from the calibration

curve.

Enzyme linked assay procedure:

All reagents are brought to room temperature before use

• Wash solution was prepared by pouring the entire contents of the

wash concentrate (50ml) into a clean volumetric flask and diluted

by adding 1200ml of distilled water (25 fold dilution) to give a

buffered wash solution.

• Calibrators were reconstituted immediately before use. Stoppers

we removed and placed inverted on a clean surface. 0.75ml exactly

of distilled water were added to each vial and the stoppers are

placed. Vials were mixed gently then allowed to stand at room

temp for at least 15 minutes to reconstitute.

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Calibrator µg/L

A 0

B 1

C 5

D 24

E 50

• Antibody solution was prepared by pouring the contents of the

tracer, HRP Anti-SCC into the vial of Biotin Anti-SCC and mixed

gently.

• The streptavidin coated microtiter plates were washed once with

the wash solution then marked to be able to clearly identify the

samples during and after the assay.

• 25 µL of the SCC calibrators (A, B, C, D, E) or patient samples

were pipetted into the bottom of the strips wells.

• 100 µL of antibody solution were added to each well using a 100

µL precision pipette.

• The frame containing the strips was covered with a plate sealer and

incubated for 1 hour at room temperature (20 – 25°c) with constant

shaking of the plate using a microplate shaker.

• Contents were aspirated or decanted from each well and washed by

adding 400 µL of wash solution per well. The process was repeated

for 5 times for a total of 6 washes. After the last wash, the contents

were aspirated or decanted. Any remaining wash solution was

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removed by tapping the inverted plate firmly on clean paper

toweling.

• 100 µL of TMB HRP substrate were added to each well as quickly

as possible and the time between the addition to the first and last

well did not exceed 5 minutes.

• The plate was covered with a new plate sealer and was incubated at

room temperature with constant shaking for 30 min.

• 100 µL stop solution were added to each well.

The optical density of each well was determined within 15 minutes using

a microplate spectrophotometer set at 405nm.

The assay procedure was performed on Technology INC – DPC

(Diagnostic products cooperation, 15700 west 96m street Los Angles,

CA90045 – 5597) (Duley et al., 1985).

Calculation of results:

The mean absorbance values for set of duplicate standards were

calculated.

The calibration curve was constructed by plotting the absorbance

(A) values obtained for each SCC calibrator against the corresponding

SCC concentration (in µg/L).

The concentration of each unknown sample was determined by

calculating the concentration of SCC corresponding to the mean

absorbance from the calibration curve.

A standard curve was run with each assay.

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Detection limit

The typical detection limit of the assay of CanAg SCC EIA is < 0.3

µg/L. The detection limit was calculated as te concentration which is 2

standard deviations above the mean zero standard.

Expected values:

The mean value was 0.58 µg/L

Range 0.16 – 1.5 µg/L

Statistical Methods:

Results obtained were analyzed; data were summarized as mean

and standard deviation and compared using t-test in comparing between

two groups and analysis of variance (ANOVA) in comparing more than

two groups. Significant results were followed up by Bonferroni post hoc

test, non-Gaussian data were summarized as medians with interquartile

range (25th – 75th percentile) and they were then log-transformed to

confirm normal distribution.

Quantitative data were compared using spearman rho correlation

(rs). The optimal cut-off for different analytes were calculated by

constructing a receiver operating characteristic (ROC) curve and odds

ratio were calculated for each parameter at the selected cuto-ff value.

A P value less than 0.05 was considered statistically significant.

Calculations of data were done on SPSS (SPSS incorporation, Chicago,

Illinois).

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Results

Results

Demographic and clinical characteristics:

This study included 65 patients with hepatic focal lesion accompanied with

liver cirrhosis (Group I). Group I was further subdivided into group Ia : 49 patients

(75%) with proved hepatocellular carcinoma and Group Ib : 16 patients (25%) with

only hepatic cirrhosis according to their histopathological findings (figure:8).

Twenty healthy subjects were included in the study to serve as controls (group II).

25%

75%

Group Ib

Group Ia

Figure (8): Histopathological findings of patient's group.

Group I included 65 patients, they were 42 males (64.7%) and 23 females

(35.3%) with male : female ratio 1.8 : 1. their ages ranging from 42 – 70 years

(60.6 + 11.28). Group II included 20 subjects, they were 12 males (60%) and 8

females (40%). Their ages ranged from 38 – 64 years (53.7 + 5.80) (tables: 8,9 and

figures: 9,10).

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Results

Table: 8

Gender distribution of subjects in the studied group.

Group Number of

males %

Number of

females %

I 42 64.7% 23 35.3%

II 12 60% 8 40%

MalesMales

FemalesFemales

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

Group I Group II

Figure : 9

Gender distribution of subjects in the studied group.

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Results

(Table : 9) Age of subjects among the studied groups.

Group Range in years Mean + SD

I 42 – 70 yrs 60.7 + 11.28

II 38 – 64 yrs 53.7 + 5.80

0

10

20

30

40

50

60

70

Group I

Age (Years)

Group II

(Figure : 10) Age distribution among the studied groups.

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Results

Pugh's modification of child classification of the cirrhotic patients (Group I)

showed that :

In group Ia: 10 patients (20.4%) of them were in score A, 19 patients

(38.7%) were in score B, while score C constituted 20 patients (40.8%).

In group Ib : 5 patients (31.3%) of them were in score A, 2 patients (12.5%)

were in score B, while score C constituted 9 patients (37.5%) (table: 10, figure 11)

(Table: 10) Child-Pugh score of the patients' group.

Item Group Ia (n = 49) Group Ib (n = 16)

Child-Pugh score

A 10 patients 5 patients

B 19 patients 2 patients

C 20 patients 9 patients

A

A

B

B

C

C

0

5

10

15

20

25

Group Ia

ABC

Child-PughScore

Group Ib

Number of patients

(Figure: 11) Child-Pugh score of the patients' group

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Results

Sonographic findings:

Among the 65 patients of the study who were presented with hepatic focal

lesion, 46 patients (71%) had solitary hepatic lesion and 19 patients (29%) had

multiple hepatic lesions (figure: 12).

71%

29%

Solitary FocalLesion

Multiple FocalLesions

(Figure: 12) Number of hepatic focal lesions in the patients groups.

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Results

Considering the lesion size of 3cm, 37 patients (57%) had lesions of less than

3cm (<3cm), while 28 patients (43%) had lesions of more than 3cm (>3cm) in size.

(figure: 13)

57%

43%

Lesion Size<3.0 cm

Lesion Size>3.0 cm

(Figure: 13) lesion size of hepatic focal lesion patients

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Results

Laboratory Data:

Regarding cirrhosis etiology, all HCC patients had chronic viral hepatitis as

proved by hepatitis markers. There were 16 patients (24.6%) due to HBV infection,

37 patients had HCV (56.9%) and 12 patients (18.4%) had mixed HBV and HCV

infection. In group Ia (HCC proved), 27 (55%) patients had HCV, 10 (20.4%)

patients had HBV and 12 (24%) patients had both HBV and HCV. In group Ib

(cirrhosis only), 10 (62.5%) patients had HCV and 6 (37.5%) patient had HBV

infection. (table: 11 figure: 14).

Table 11: The etiology of cirrhosis in group I patients

Item Group Ia (n = 49) Group Ib (n = 16)

Cirrhosis etiology

HCV

HBV

HCV & HBV

27 (55%)

10 (20.4)

12 (24%)

10 (62.5%)

6 (37.5%)

None (0%)

27

10

12

610

Group IbHCV

Group IbHBV

Group IaHCV

Group IaHCV&HB

Group IaHBV

(Figure: 14) Etiology of cirrhosis in group I patients

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Results

Concerning liver functions: the mean values of ALT (U/L) and AST (U/L)

respectively in group Ia were: 38.27+6.32 U/L and 55.45+4.21 U/L while the mean

values for ALT and AST for group Ib patients were 42.6+4.28 and 59.73+3.67

respectively. All group II subjects had normal level of ALT, mean value was

14.9+2.11 and AST mean value was 23.85+5.70. Serum albumin levels in g/dl were

as follows: mean value for group Ia was 2.44+0.1, mean value for group Ib was

3.42+0.23 while for normal controls means value was 3.9+1.02. (table: 12, figures

15 & 16)

Table 12: The laboratory results of group Ia, group Ib and group II.

Laboratory results Group Ia Group Ib Group II

ALT (U/L) 38.27+6.32 42.6+4.28 14.9+2.11

AST (U/L) 55.45+4.21 59.73+3.67 23.85+5.70

Albumin (g/dl) 2.44+0.1 3.42+0.23 3.9+1.02

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Results

ALT

ALT

ALT

AST

AST

AST

0

10

20

30

40

50

60

70

Group Ia

ALTAST

Group Ib Group II

u/L

(Figure: 15) ALT and AST test results in group I and group II

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

Group Ia

Albumin

Group Ib Group II

g/dl

(Figure: 16) Albumin level in group I and group II.

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Results

The values of AFP and SCC-Ag were not normally distributed so that the

median level was calculated.

Concerning AFP:

The Median level for Alpha fetoprotein in group Ia, group Ib and group II

was 290+947.6, 67.50+247.26 and 4.40+6.8 respectively (table 13). P value

between group Ia and group Ib was 0.001 so there was high statistically significant

difference between the 2 groups. Statistical analysis of p value between group Ia

and group II was <0.0005 (high statistically significant difference) also the

difference between group Ib and group II was statistically significant (P = 0.003).

(Figure: 17).

Table 13: The level of AFP in group Ia, group Ib and group II

Item Group Ia Group Ib Group II

Median level 290+947.6 67.50+247.26 4.40+6.8

0

50

100

150

200

250

300

350

Group Ia Group Ib Group II

AFP(IU/ml)

(Figure: 17) The Median level of AFP in the studied groups Ia, Ib, II

100

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Results

Concerning SCC-Ag:

The Median level for SCC-Ag in group Ia, group Ib and group II was

2.84+0.5, 1.52+0.37 and 0.7+0.29 respectively, (Table 14). P value between group

Ia and group Ib was 0.001 so there was high statistically significant difference

between the two groups. Statistical analysis of P value between group Ia and group

II was <0.0005 (high statistically significant difference) while the difference

between group Ib and group II was statistically significant. (P = 0.043) (Figure 18).

Table 14: The level of SCC-Ag in group Ia, group Ib and group II

Item Group Ia Group Ib Group II

Median level 2.84+0.5 1.52+0.37 0.7+0.29

0

0.5

1

1.5

2

2.5

3

Group Ia Group Ib Group II

Scc-Ag(ug/L)

(Figure: 18) The level of SCC-Ag in group Ia, group Ib and group II.

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Results

When using the Receiver Operating Characteristic (ROC) curve to determine

the best specificity and sensitivity of both analytes, at the value of cutoff 40 IU/ml,

the specificity and sensitivity of Alpha fetoprotein were 100% and 67.2%

respectively. While at the value of cutoff 2.55 µg/L, the specificity and sensitivity

of SCC-Ag were 100% and 61.2% respectively. The area under the curve (AUC)

for Alpha fetoprotein was 0.859 with a confidence interval ranging from 0.776 –

0.942 while that of SCC-Ag was 0.788 with a confidence interval ranging from

0.689 – 0.886. (Figure: 19).

(Figure 19): ROC analysis of Alpha fetoprotein (AFP) and Squamous cell

carcinoma antigen (SCC-Ag)

Using different cutoff values as reported in different literatures, showed that

at a cutoff of serum Alpha fetoprotein 200 IU/ml, the sensitivity was 35% and the

specificity was 100% while at a cutoff >400 IU/ml, the sensitivity decreased to

7.6%. At cutoff of serum SCC-Ag 1.2 (the kit cutoff) the sensitivity was 65.2% and

the specificity was 36.7%.

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Results

When both AFP and SCC-Ag were measured together, the sensitivity was

calculated at the best-chosen cutoff values, and sensitivity was improved to 87.7%

with specificity of 100%. (Table 15).

Table (15): Combined sensitivity and specificity for AFP and SCC-Ag.

Alpha fetoprotein SCC-Ag

Cirrhosis (<40 IU/ml) HCC (>40 IU/ml) Total

Cirrhosis (<2.55 µg/L) 22 16 38

HCC (>2.55 µg/L) 10 17 27

Total 32 33 65

+ve AFP /+ve SCC-Ag = 17/49.

+ve AFP /-ve SCC-Ag = 16/49.

-ve AFP /+ve SCC-Ag = 10/49.

Total = 43/49 = 87.7%.

When the lesion size (using 3cm value) and the lesion number (single or

multiple) were correlated with the level of Alpha fetoprotein (40 IU/ml) and SCC-

Ag (2.55 µg/L), the only significant correlation came between Alpha fetoprotein

level and the lesion size of less than 3cm (P = 0.01).

103

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Discussion

Discussion

Hepatocellular carcinoma (HCC) is the fifth most common cancer in the

world. Because of its increased incidence in the last decade and the expected

further increase in the next 2 decades, HCC is arousing great interest.


Carcinogenesis of HCC is a multi-factor, multi-step and complex

process, which is associated with background of chronic and persistent

infection of hepatitis B virus (HBV) and/or hepatitis C virus (HCV). These

high risk patients are closely followed up, and increasing number of small

equivocal lesions are detected by imaging diagnosis. They are now widely

recognized as precursor or early stage HCC and are classified as dysplastic

nodule or early HCC. (Sakamoto et al., 2008). These infections along with

alcohol and aflatoxin B1 intake are widely recognized etiological agents in

HCC. (Yu and Keeffe, 2003).

There is considerable geographical variation in the incidence of HCC in

Egypt, HCC is third most frequent cancer in men with >8000 new cases

predicted by 2012. (Goldman et al., 2007). Up to 90% of HCC cases in the

Egyptian population were attributed to HCV. Egypt has the highest prevalence

of hepatitis C virus (HCV) in the world, apparently due to mass parenteral

antischistosomal therapy. Estimating the further burden of HCV in Egypt is

important to support health policies to combat the epidemic (Deuffic-Burban

et al., 2008). In the USA, the increasing incidence of HCC has been associated

with HCV infection. Studies of HCV progression to HCC are expected to

provide new insights on the management of this increasing problem and

therefore are of great public health interest (Goldman et al., 2007).

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Discussion

HCC is recognized for its heterogeneous clinical and biological

presentation, variable natural course, and its relationship to defined risk factors

and aetiologic agents, as well as the difficulty in predicting response to

different modes of treatment. The time interval from an undetectable tumor to a

2cm lesion may vary between four and twelve months which leaves a relatively

narrow window for optimal intervention in already established tumor with fast

doubling time. (Shouva.l, 2002).

Current diagnosis of HCC relies on clinical information, liver imaging

and measurement of serum alpha-fetoprotein (AFP). (Goldman et al., 2007).

Unfortunately, although US scanning is a powerful technique it is subjective

and operator skill dependent. On the other hand, AFP the only serological

marker currently available in clinical practice, is not a sufficiently reliable

marker to identify HCC patients, mainly because of its poor sensitivity

(Giannelli et al., 2007) and elevation in non-malignant liver disease (Li et

al.,2008). About 3 – 4% of cirrhotic patients develop primary liver cancer

every year. Specific serological markers have not yet been identified for

screening of high risk patients. Hence, there is a great need for new biomarkers.

(Teofanescu et al., 2008).

The squamous cell carcinoma antigen (SCCA), a member of the serpin

family physiologically expressed in the skin, has been reported to be over

expressed in HCC tissue and serum. (Giannelli et al., 2005) and at a lower

extent in cirrhosis and chronic hepatitis (Turato et al., 2008). Squamous cell

carcinoma antigen (SCCA) variants has been found to be remarkably

overexpressed in the liver of patients affected by hepatocellular carcinoma

(HCC), being affected in 100% of HCC surgical biopsies by

immunohistochemistry. (Beneduce et al., 2004).

105

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Discussion

This study was designed to assess the levels of SCCA and AFP in

Egyptian patients with HCC and to assess the clinical utility of SCC-Ag as a

non invasive marker for early diagnosis of HCC.

In this study, the mean age of hepatic focal lesion patients was

60.7+11.28 (range 42 – 70 years). This age is agreed upon by Giannelli et al.,

2007 who stated the mean age of the selected HCC patients for his study was

66.5+9.7 and Pontisso et al., 2004 who found the HCC patients of the studied

group had a median age of 65 years (range 23 – 76 years).

In this study the male / female ratio of hepatic focal lesion patients was

1.8 : 1 which is agreed upon by the study of Pontisso et al., 2004 in which

male/female ratio of the HCC patient where 2 : 1.

Regarding cirrhosis aetiology of the studied 65 patients with hepatic

focal lesion, in total, 37 patients were anti-HCV positive, 16 patients were

HBsAg positive and 12 were co-infected with HBV and HCV. These data

match with Pontisso et al., 2004 who investigated 65 HCC patients. In total, 37

patients also were anti-HCV positive, eight were HBsAg positive, six were co-

infected by HBV and HCV. But eight admitted alcohol abuse and the

remaining six patients had no risk factor identified.

Farinati et al., 2006. evaluated a large series of HCC patients (1.158

patients) with reference to serum AFP levels at the diagnosis. Patients were

divided into three AFP groups: Normal (20 IU/ml) [4.6%], elevated (21 – 400

IU/ml) [36%) and diagnostic (>400 IU/ml) [18%]. The authors thus confirmed

the low sensitivity (54%) of AFP in the diagnosis of HCC.

Arrieta et al., 2007 investigated 74 patients with cirrhosis without

hepatocellular carcinoma and 139 patients with hepatic lesions diagnosed by

biopsy and shown by image scans were included. Sensitivity and specificity of

106

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Discussion

determination of alpha-fetoprotein >or = 200 and 400 IU/ml were analyzed.

Areas under the curve were compared. They found that for an elevation of AFP

> or = 200 and 400 IU/ml the specificity was 100% in both cases, with a

sensitivity of 36.3% and 20.2% respectively. Similar results were found in our

study, at the cutoff of serum α-fetoprotein 200 IU/ml the sensitivity was 35%

and the specificity was 100% while at a cutoff >400 IU/ml the sensitivity

decreased to 7.6%.

Being of low sensitivity AFP is not an ideal marker for HCC diagnosis or

screening. On the other hand, SCC-Ag has a better sensitivity; in our results at

the cutoff of serum SCC-Ag 1.2 µg/L (the kit cutoff) the sensitivity was 65.2%

and the specificity was 36.75%. similar results were found by Giannelli et al.,

2005 who investigated 120 patients with HCC and 90 patients with liver

cirrhosis. Both serologic markers for HCC were measured: SCC-Ag and AFP.

The authors reported that as a marker for HCC, SCC-Ag has high sensitivity

(84.2%) but low specificity (48.9%) at the kit cut off (0.3 µg/ml).

In this study, when combined sensitivity of both markers, calculated at

the best-chosen cutoff values (SCC-Ag 2.55 µg/L and AFP 40 IU/ml)

sensitivity improved to 87.7% with specificity of 100%. Matching results were

found by Giannelli et al., 2005, although a different cutoff was used,

combining both markers increase the sensitivity, the used cutoff values were

0.3 µg/L for SCC-Ag and 20 IU/ml for AFP, there was a 90.83% correct

diagnosis rate among the HCC patients (109/120), with 44.44% (40/90) true

negatives among the cirrhotic patients. The positive predictive value was

68.55% (109/159). Although there remained a low specificity, the number of

correct HCC serologic diagnosis increased to 55.05% (60/109).

In this study, there was no significant correlation between AFP and SCC-

Ag on one hand and the tumor size (using 3cm value) and number on the other

107

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Discussion

hand. The only significant correlation came between AFP level and the lesion

size of less than 3cm, similar results were reported by Giannelli et al., 2005,

who found no significant correlation between serum level of SCC-Ag and the

tumor size. On the other hand Pisit et al., 2000 who studied 309 pathologically

proven HCC cases divided into three groups: group 1 with normal AFP (<20

IU/ml), group 2 with moderately elevated AFP (20 – 399 IU/ml) and group 3

with markedly elevated AFP (>400 IU/ml). The authors found that HCC

patients with high AFP tended to have greater tumor size, bilobar involvement,

massive or diffuse types, and portal vein thrombosis, but Nakao et al 1998

found no significant correlation between serum level of AFP and the tumor

size.

108

EG1200352

Summary and Conclusion

Summary and conclusion

Hepatocellular carcinoma (HCC) is the fifth most common cancer

in the world. Because of its increased incidence in the last decade and the

expected further increase in the next two decades, HCC is arousing great

interest.

Rapid detection and early treatment are important to improve the

prognosis of this aggressive tumor as once symptoms appear most of the

tumors will be unresectable.

Surveillance of high-risk individuals for HCC is commonly

performed using the serum marker AFP often in combination with

ultrasonography.

For decades, AFP was the only tumor marker for detection of

HCC. The serum concentration of 20 IU/ml is the most commonly used

cut-off value for AFP to differentiate HCC patients from healthy adults in

clinical researches. AFP specificity is poor when levels fall within the

"grey area" i.e. 10 – 500 IU/ml. There is thus a final need to increase the

specificity of AFP.

It was reported that the best method to detect HCC is the

simultaneous measurement of more than one marker to improve

sensitivity and specificity of testing.

This study was done to assess the clinical utility of SCC-Ag as a

non invasive marker in the early diagnosis of HCC.

This study was conducted on 65 newly diagnosed hepatic focal

lesion cases receiving no treatment from those attending the Tropical

Clinic in Cairo University Hospitals (Group I) as well as 20 age and sex

109

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Summary and Conclusion

matched healthy control subjects (Group II). Group I was further

subdivided into group Ia (HCC proved patients) and group Ib (cirrhosis

only) according to their histopathological findings. All patients were

subjected to full history taking, clinical examination, abdominal CT and

pathological examination.

Laboratory tests included routine investigations for HBV (HBs Ag,

HBc Ab) and HCV (HCV antibodies), ALT, AST, and serum albumin.

Specific laboratory investigations included serum AFP by

immunoradiometric assay (IRMA) and SCC-Ag by enzyme linked

immunosorbent assay (EIA).

Median levels of serum AFP and SCC-Ag in group Ia was

significantly higher when compared with both the group Ib and group II

(P<0.0005 for both of them). On using the receiver operator characteristic

(ROC) curve to improve the specificity and sensitivity of AFP and SCC-

Ag, the cut-off value of 40 IU/mL and 2.55 µg/L yielded a sensitivity of

67.2% and 61.2% respectively and specificity of 100% (best cutoff). The

diagnostic sensitivity of them was increased to 87.7% when they were

calculated together. The level of AFP showed significant correlation with

the lesion size less than 3cm (p=0.01).

Simultaneous use of AFP and SCC-Ag in the screening of patients

with hepatic focal lesions may increase the chance of early diagnosis of

HCC patients.

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RECOMMENDATIONS Further studies are recommended to:

1- Asses the serum levels of SCCA in a large number of HCC Egyptian patients.

2- Follow up of patients who underwent tumor

resection to correlate between AFP and SCCA and combine it with liver imaging and pathological examination for early detection of tumor recurrence.

3- In order to increase the diagnostic

significance of AFP, AFP isoforms should be identified and studied as they are helpful in differentiating between benign and malignant lesions of the liver.

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